Small molecule dual-inhibitors of TRPV4 and TRPA1 for sanitizing and anesthetizing

ABSTRACT

Provided are methods of sanitizing a subject, and methods of anesthetizing a subject. Further provided are methods of treating and/or preventing dermatological disorders, reducing skin inflammation, reducing pain, and/or reducing itch in a subject in need thereof. The methods may include administering to the subject an effective amount of a TRPA1 and/or TRPV4 inhibitor. Further provided are compositions including a TRPA1 and/or TRPV4 inhibitor compound in combination with a carrier, vehicle, or diluent that is suitable for topical application.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. national stage entry, under 35 U.S.C. 371,of international application number PCT/US2017/026714, filed Apr. 7,2017, which claims priority to U.S. Provisional Patent Application No.62/319,684, filed Apr. 7, 2016, U.S. Provisional Patent Application No.62/331,951, filed May 4, 2016, and U.S. Provisional Patent ApplicationNo. 62/337,701, filed May 17, 2016, each of which is incorporated hereinby reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersDE018549 and DE01852951 awarded by the National Institutes ofHealth/National Institute of Dental and Craniofacial Research(NIH/NIDCR), and grant numbers AR059402, AR31737, and AR050452 awardedby the National Institutes of Health/National Institute of Arthritis andMusculoskeletal and Skin Diseases (NIH/NIAMS). The government hascertain rights in the invention.

SEQUENCE LISTING

The sequence listing is filed with the application in electronic formatonly and is incorporated by reference herein. The sequence listing textfile “028193-9128-US12_As_filed_Sequence_Listing” was created on Oct. 4,2018 and is 776 bytes in size.

FIELD

This disclosure relates to methods and compositions for sanitizing asurface, anesthetizing a subject, and treating inflammation, pain, itch,cancer, autoimmune diseases, fibrotic diseases, skin pigmentation,and/or other dermatological disorders.

INTRODUCTION

The skin is the largest organ in many vertebrates, including humans. Itprovides barrier protection against the potentially harmful externalenvironment. The skin also represents the site of first interaction ofthe ambient environment to immunologically competent and sentientstructures of the organism. Cells endowed with sensory transductioncapacity for warmth, cold, mechanical cues, pain, and itch are sensoryneurons in the dorsal root and trigeminal ganglia with their peripheralaxons directly interfacing with skin. However, successfully targetingthe skin for treatment of inflammation, pain, itch, cancer, autoimmunediseases, fibrotic diseases, skin pigmentation, and other dermatologicaldisorders has remained elusive.

Biochemical pathways in the skin include those relating to the transientreceptor potential (TRP) superfamily of ion channels. One ion channel inthis family is TRPV4. TRPV4 is a multimodally-activated non-selectivecation channel permeable to calcium (i.e., Ca++). The TRPV4 ion channelis expressed robustly in epidermal keratinocytes of mammalian skin.However, TRPV4 is also expressed in skin-innervating sensory neurons. InTrpv4−/− mice, an epidermal phenotype of impaired barrier functionbetween epidermis and dermis has been shown. In regards to painsignaling, TRPV4 has been found critical for physiological withdrawalresponses to noxious osmotic and mechanical, but not thermal, cues andhas also been found relevant for inflammation or nerve-damage-inducedsensitization of nociception. While it is understood that TRPV4 isexpressed in epidermal keratinocytes and skin-innervating sensoryneurons, an in vivo role of TRPV4 in pathological pain evoked by UVBexposure has not been demonstrated. Moreover, a direct role of TRPV4 initch transmission has not been demonstrated as of yet.

TRPA1 is another TRP ion channel located on the plasma membrane. TRPA1acts as sensor for environmental irritants, pain, cold, and stretch.Although TRPV4 and TRPA1 function in the skin, it is not known whethertargeting TRPV4 and/or TRPA1 would be useful in the treatment ofinflammation, pain, itch, cancer, autoimmune diseases, fibroticdiseases, skin pigmentation, and other dermatological disorders.Furthermore, specific TRPV4 and TRPA1 inhibitors are not presentlyknown. New and successful treatments for dermatological disorders, aswell as and methods for sanitizing and anesthetizing are needed.

SUMMARY

In an aspect, the disclosure relates to a composition comprising aninhibitor and iodine, wherein the inhibitor inhibits TRPV4, TRPA1, or acombination thereof.

In an aspect, the disclosure relates to composition comprising aninhibitor and an anesthetic, wherein the inhibitor inhibits TRPV4,TRPA1, or a combination thereof.

In an aspect, the disclosure relates to an inhibitor that inhibitsTRPV4, TRPA1, or a combination thereof.

In some embodiments, the inhibitor does not inhibit TRPV1, TRPV2, orTRPV3. In some embodiments, the inhibitor comprises a compound accordingto Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring. In someembodiments, the inhibitor comprises a compound selected from thefollowing:

In another aspect, the disclosure relates to methods of sanitizing asubject. In some embodiments, the method includes contacting the subjectwith the composition as detailed herein. In some embodiments, thesubject is contacted with the composition for a period of timesufficient to cause a reduction in the population of microorganisms onthe subject. In some embodiments, the composition is administered to asurface of the subject, wherein the surface is selected from the groupconsisting of a skin area, a wound, and an ulcer. In some embodiments,the composition disinfects the subject. In some embodiments, thecomposition sterilizes the subject. In some embodiments, the compositionhas antibacterial activity.

In another aspect, the disclosure relates to methods of anesthetizing asubject. In some embodiments, the method includes administering to thesubject the composition as detailed herein. In some embodiments, themethod includes co-administering an anesthetic and an inhibitor to thesubject, wherein the inhibitor inhibits TRPV4, TRPA1, or a combinationthereof. In some embodiments, the composition sanitizes and reducespain.

In another aspect, the disclosure relates to methods of treating and/orpreventing a dermatological disorder in a subject in need thereof, themethod comprising administering to the subject an effective amount of aTRPV4 and/or TRPA1 inhibitor. In some embodiments, the dermatologicaldisorder is selected from the group consisting of pancreatitis,epilepsy, arthritis, osteoarthritis, multiple sclerosis, stroke, CNSautoimmune condition, traumatic brain injury, spinal cord injury, brainedema, CNS infection, neuro-psychiatric disorder, skeletaldegenerative-inflammatory disorder, trigeminal pain, colitis, andsclerosis. In some embodiments, the trigeminal pain comprises headache.

In some embodiments, the TRPV4 and/or TRPV4 inhibitor comprises acompound according to Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring. In someembodiments, TRPV4 and/or TRPA1 inhibitor comprises a compound selectedfrom the following:

In some embodiments, the compound inhibits TRPV4 and TRPA1. In someembodiments, the compound does not inhibit TRPV1, TRPV2, or TRPV3.

The disclosure provides for other aspects and embodiments that will beapparent in light of the following detailed description and accompanyingFigures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1G: Keratinocyte-specific and inducible Trpv4 null mouse andits UVB response. (FIG. 1A) Gene-targeting of Trpv4 and geneticmanipulation underlying generation of keratinocyte-specific andinducible Trpv4 knockout mice. Shown are sequential steps of mouse Trpv4targeting, starting with flanking Trvp4 exon13 with loxP elements andinsertion of a selection cassette, flanked by frt sites, in mouseembryonic stem cells. After generation of chimeric mice and stabletransmission of the engineered mutation, the selection cassette wasremoved by breeding to FLPe mice. Resulting mice were homozygosed andcrossed with K14-CRE-ER^(tam) mice, which then permittedkeratinocyte-specific and inducible Trpv4 knockout/knockdown. (FIG. 1B)DNA genotyping. Shown are PCR products of WT, heterozygote andhomozygous Trpv4^(lox/lox) mice. Note that the PCR products needed to bedigested with Pacl, and that all mice were pre-screened to be CRE+ byanother genotyping PCR. (FIG. 1C) Co-labeling of mouse skin forkeratin-1 and keratin-14 indicate the established pattern forvehicle-induced control mice (upper panel), and a similar pattern forspecific TRPV4 knockdown in keratinocytes (lower panel). However, inthese animals note a slightly increased expression of K14 in the stratumspinosum, reflecting attenuated TRPV4 expression and thus reduced Ca⁺⁺influx. K14 is normally down-regulated at the basal-to-suprabasaltransition, concomitant with the rise in Ca⁺⁺-signaling and induction ofterminal differentiation. (FIG. 1D) TRPV4 protein expression in L5 DRGneurons not different between genotypes. Densitometry of TRPV4immunohistochemistry in L5 (=foot-pad innervating) DRG neurons (upperpanel micrographs), the bar-diagram illustrates the lack of a differencein terms of TRPV4 protein abundance in oil- vs. tam-treated mice, forboth base-line and 48 hours after UV exposure, confirming thespecificity of TRPV4 knockdown in skin when using K14 as CRE driver.Note the characteristic morphology of decorated cells identifying themas DRG sensory neurons. Note also the different levels of TRPV4expression in these neurons, as noted previously; n=3 mice/group, 50neurons/mouse. (FIG. 1E) Lack of TRPV4 expression in Merkel cells infoot-pad epidermis. A confocal triple-fluorescent micrograph panel isshown, depicting representative images of immuno-labeled paw-pads fromiKO control vs. tamoxifen-induced mice. Note complete knockdown of TRPV4in this example (red channel). For Merkel cells (green channel), ananti-cytokeratin 8 antibody was used. Note lack of TRPV4 co-labeling inMerkel cells. Blue channel=DAPI. (FIG. 1F) Lack of effect of tamoxifenapplication in K14-CRE-ER^(tam) mice on UVB behavioral sensitization.Note very similar withdrawal thresholds in (K14-CRE-ER^(tam) XTrpv4^(lox/+)) mice (=Trpv4 heterozygotes in keratinocytes when inducedwith tamoxifen) for noxious mechanical (upper diagram) and thermal(lower diagram) stimulation; n=7 mice per group. Also note thetime-course with peak sensitivity at time-point 48 hours. (FIG. 1G) Sizedistribution of pERK-expressing L5 DRG neurons in oil-treated iKO mice,exposed to UVB. The bar diagram illustrates size prevalence of small andmedium-size sensory neurons that express pERK 48 hours after UVBexposure, note absence of larger neurons (>1200 μm²), n=22 neurons.

FIGS. 2A-2G: UVB stimulation device and UVB keratinocyte controlexperiments. (FIG. 2A) UV spectrum emitted by the LEDs, overlapped withthe spectrum of quartz (red trace), which is almost fully permeable toUVB, and glass (blue trace), which has a very low UVB permeability.(FIG. 2B) Focusing properties of the ball lens. (FIG. 2C) Focal geometryof the combination of UV-LED and ball lens. (FIG. 2D) Absence of thermaleffects of the UV-LEDs; measurement of temperature in the focal pointover time. (FIG. 2E) TRPV3 activation experiment. Induction of a Ca++transient by camphor, which can be blocked effectively by 10 μM IPP,suggesting TRPV3-mediated signaling. (FIG. 2F) TRPV4 selective activatorGSK101-related findings. Ca++ transient in 1° MK in response to 5 nMGSK101, which can be completely blocked by 20 μM GSK205, suggesting itis specifically mediated by TRPV4. The GSK101-response can also beeliminated by absence of external Ca++, in keeping with TRPV4 signaling.(FIG. 2G) TRPV4 is sufficient for the UVB-Ca++ response—HEK293T cellheterologous transfection. Directed expression of TRPV4 in HEK293T cellsleads to a Ca++-transient in response to UVB radiation, which is greatlyreduced in control-transfected cells. Preexposure to 20 μM GSK205virtually eliminates the Ca++-signal in TRPV4-transfected cells, andeliminates the moderate signal of control-transfected cells.

FIGS. 3A-3E: Keratinocyte-specific ablation of Trpv4 leads toalterations in nocifensive behavior in response to UVB. (FIG. 3A)Epidermal TRPV4 expression and its loss upon keratinocyte-specificablation of Trpv4 in tam-induced iKO mice. (i) TRPV4 immunofluorescence.Note TRPV4 in epidermis of vehicle (oil) treated control, but nottam-induced iKO mice. Bar=10 μm. (ii) Western blot of epidermal lysatesfrom paw-pad skin. Note knockdown and more complete loss of TRPV4following induced Trpv4-ablation (β-actin used for normalization). (iii)qRT-PCR for Trpv4 mRNA from paw-pad skin is shown, indicatingsignificant Trpv4 knockdown in response to tam-treatment vs. carrier(oil). P<0.0001, t-test. (iv) Immunofluorescence for epidermal lineagemarkers. In WT skin, basal epidermal marker keratin-14 is downregulatedand suprabasal marker keratin-1 is induced upon commitment to terminaldifferentiation. Upon knockdown of TRPV4, this balance appearsperturbed, with some spinous layer cells showing co-labeling. Bar=10 μm.(FIG. 3B) Nocifensive behavior in response to UVB exposure. Time-course(in hours) for nocifensive behavior elicited by either a noxiousmechanical stimulus (automatic von Frey hair assay, left) orthermally-evoked nocifensive behavior (Hargreaves' assay, right). Notesignificantly less sensitization in Trpv4^(−/−) and in tam-treated iKOmice, relative to oil-treated (vehicle) iKO and WT mice. n≥10 animalsper group; **p<0.01 ANOVA. (FIG. 3C) Correlation between nocifensivebehavior and level of Trpv4 knockdown. n=12 animals are shown for whichboth parameters were available and Trpv4 mRNA levels <0.45. Note thefour vehicle-induced animals (green symbols) vs. their tamoxifen-inducedcounterparts (red symbols). (FIG. 3D) Loss of epidermal TRPV4 shows nosignificant effect on nocifensive behaviors caused by formalininjection. Bars depict average cumulative nocifensive behavior withinthe first 10 minutes (phase I), and 10-45 minutes (phase II)post-injection. n=4 per group. (FIG. 3E) Phosphorylated ERK in L5 DRGneurons. pERK immunofluorescence of L5 DRG sections are shown for oil-and tam-treated iKO animals±exposure to UVB. Note that only UVB-exposedcontrol mice show pERK expression in the paw-pad-innervating L5 DRG.Quantifications are shown at right. n=3 animals per group, 6 sectionsper DRG per animal, **p<0.01 ANOVA.

FIGS. 4A-4E: Structural and ultrastructural analyses showing thatUVB-mediated skin tissue injury depends upon keratinocyte TRPV4, andImmuno-histochemical analysis demonstrates that UVB-mediated activationof keratinocytes and recruitment of macrophages and neutrophils dependsupon keratinocyte TRPV4. (FIG. 4A) 1 μm toluidine-blue semi-thinsections. Micrographs show representative findings of skin in responseto UVB, sampled 48 hours after UVB exposure. Note that upon UVBstimulation, oil-(TRPV+) but not tam-treated (TRPV−) iKO mice exhibitseparations at the epidermal-dermal boundary and robust signs of tissueinjury; note granulocytes (Gr, neutrophil). Note also that just beneaththe stratum corneum (SC), the upper epidermis shows extensive structuraldamage which could also be seen in skin of tam-treated iKO mice whereTrpv4 knockdown was incomplete, but not in those animals where it wasmore complete (see FIG. S2A). Bars=20 μm. Der=dermis; Epi=epidermis.(FIG. 4B) Ultrastructural findings by EM. Selected areas from 1 μmsemithin sections of paw skin were examined by transmission electronmicroscopy. (A-A′) and (C-C′) show normal epidermal (Epi) structure forboth, oil- and tam-treated iKO mice, in the absence of UVB stimulation.(A) and (C) show and intact epidermis. Basal (BL) and spinous (Sp)layers are magnified A′ and C′ displaying a normal organization with noevidence of epidermal damage. (B,B′B″), (D-D′) and (E-E′) showrepresentative findings of skin in response to UVB, sampled 48 h afterUVB exposure. (B) Disrupted epidermis in oil treated iKO mice. An areaequivalent to the boxed area is magnified in (B′), where granulocyteinfiltration of epidermis is evident (Gr) and blistering with detachmentof the epidermis from the dermis (double arrows). (B″) Upper part ofepidermis in contact with stratum corneum (SC), showing extensivevacuolization and deposits of fibrin inside the vacuoles (asterisks).(D) Tamoxifen treated iKO mice with incomplete knockdown of trvp4 showsimilar skin phenotype to oil treated iKO mice, with robust signs oftissue damage to basal and spinous layer, fibrin deposits (asterisks)and intercellular spaces (arrowheads in D′). (E) Intact epidermis in iKOwith complete knockdown of trvp4, with normal basal and spinous layersin (E′). Der, dermis. Dotted lines indicate the dermo-epidermalboundary. Bars=20 μm for A, B, C and D; 10 μm for B′ and E′ and 2 μm forthe other micrographs. (FIG. 4C) IL-6 upregulation in keratinocytes asmarker of epidermal activation. IL-6 immunofluorescence reveals areduced ability of TRPV4-deficient mice to elevate keratinocyte IL-6expression in response to UVB exposure. Quantifications for protein isshown next to micrograph. Densitometries are for n≥3 mice per group,showing significant upregulation for oil-treated iKO mice, lack thereoffor tam-treated. Right-hand bar diagram shows Il-6 mRNA quantificationand time-course. Il-6 mRNA was determined by qPCR after isolation oftotal RNA from paw-pad epidermis. Note the early and robust increase,albeit with variation, at the 2 hour time-point, in WT controlepidermis, in contrast the very moderate increase in Trpv4^(−/−)epidermis. Note also the sustained robust upregulation at 24 hours,again moderately upregulated in Trpv4^(−/−) epidermis. Quantificationsare for n=8-12 mice/group. * denotes statistically significant (p=0.011,t-test); scale-bar=20 μm. (FIG. 4D) Recruitment of macrophages inUVB-exposed skin. Note that the numbers of dermal CD68+ macrophagesinduced by UVB-exposure in control mice is significantly reduced whenTrpv4 is ablated in the epidermis. Quantifications are shown at right(n=3 mice/group; *p<0.05 t-test); scale-bar=20 μm. (FIG. 4E) Recruitmentof elastase-expressing neutrophils to UVB-exposed skin. Shown arerepresentative immunofluorescence micrographs and respectivequantifications. Note a strong increase in abundance ofelastase-expressing neutrophils in control mice, and a lack thereof intam-treated iKO mice. (n=4 mice/group, *p<0.05 t-test); scale-bar=40 μm.

FIGS. 5A-5E: Histopathology in Trpv4−/− and control mice in response toUVB. (FIG. 5A) Trpv4 knockdown level of samples shown in FIGS. 4A-4E.This bar diagram shows relative level of knockdown of Trpv4 incomparison with WT, of UVB-exposed skin samples shown in FIG. 4 . Anadjacent sample of hindpaw skin was RNA-extracted at 48 h post-exposureand subjected to Trpv4 qRT-PCR; pooled WT mRNA values from 10 mice wereset as 100%. (FIG. 5B) Light microscopic analyses of 1 μm semithinsections findings from Trpv4−/− and WT control mice. Normal skin isshown in the upper row for both genotypes in the unstimulated state,presence of epidermal and dermal inflammation in WT control vs. absencethereof in Trpv4−/− when exposing the skin to UVB, sampling conducted at48 hours. Note inflammatory changes similar to those of oil-treated iKOmice, as shown in FIG. 4 . (FIG. 5C) Ultrastructural analyses ofTrpv4−/− and WT control mice. (A-A′) and (B-B′) WT and Trpv4−/− miceshow normal skin morphology with intact epidermis (Epi) in the absenceof UVB stimulation. A′ and B′ show higher magnification of basal layer(BL) cells. (C-C′) Damaged epidermis with vacuolization (inset in C) andgranulocyte (neutrophil) (granulocyte—Gr) infiltrate (C′). (D-D′) Normalepidermal and dermal ultrastructure in Trpv4−/− mice exposed to UVB.Der-dermis; NT, nerve terminals. Dotted lines indicate thedermo-epidermal boundary. Bars=10 μm for A, B, C, C′ and D and 2 μm forthe other micrographs. (FIG. 5D) IL-6 upregulation in epidermalkeratinocytes in response to UVB depends on Trpv4; findings fromTrpv4−/− and WT control mice. Fluorescent micrographs from Trpv4−/− andWT control skin, unexposed and exposed to UVB are shown. Note strongIL-6 signal in WT, exposed to UVB, and low signal in Trpv4−/− for bothnon-exposed and UVB-exposed states. Also note IL-6-expressinginnervating peripheral nerve endings in the dermis. (FIG. 5E) Nodifference in mast cell abundance in UVB-photodermatitis in iKO mice.Left-hand micrograph shows mast-cells within sub-epidermal inflammatorytissue, stained with toluidine-blue, in an iKO mouse induced withtamoxifen, right micrograph its counterpart in an oil-treated iKO mouse.Mast-cells are indicated by white arrow-heads. Bar=20 μm. Right-hand bardiagram indicates quantification of mast-cell count per 63× visual field(5 fields per mouse, 3 mice per group).

FIGS. 6A-6H: Ca++ influx into keratinocytes in response to UVB dependson TRPV4. (FIG. 6A) Custom-built UVB cell illumination apparatus. Seealso FIG. 2 . (FIG. 6B) Fluo-4 Ca++ imaging in 1° MKs. Fluorescentmicrographs of 1° MKs after loading with Ca++-sensitive dye, fluo-4,before (upper) and at the end of UVB exposure (lower). Bar=10 μm. C—HUVB-evoked Ca++ signaling profiles. Fluo-4 imaging was used to detectCa++ transients in 1° MKs following UVB exposure. y-axis indicates theincrease in fluorescence, ΔF, normalized for prestimulation signal, F0(ΔF/F0). The signal shown is that averaged from ≥50 cells. (FIG. 6C)Ca++ signaling is dependent upon UVB, and is strikingly reduced whenquartz coverslips are replaced by glass ones, which prevent UVBpermeation (see FIG. 2A). Note that this particular Ca++ signal in WT 1°MKs persisted after UVB, as is sometimes observed. (FIG. 6D) UVB-evokedCa++ signaling is dependent on external [Ca++]. (FIG. 6E) UVB-evokedCa++ signaling is not seen in Trpv4−/− 1° MKs, revealing the importanceof the TRPV4 ion channel. (FIG. 6F) UVB-evoked Ca++ signaling isstrongly down-regulated in the presence of TRPV4-selective inhibitor,GSK205 (20 μM). (FIG. 6G) The UVB-evoked Ca++ signal is not inhibited bythe TRPV3-selective inhibitor, IPP. For validation of IPP's activity,see FIG. 2E. (FIG. 6H) The UVB-evoked Ca++ signal can be stronglyreduced with specific PLC inhibitor, U73122.

FIGS. 7A-7D: Central role for keratinocyte TRPV4 in UVB-evoked Ca++signaling and nocifensive behavior—effects of ET1. (FIG. 7A) Effects ofET1 on UVB-evoked Ca++ signaling in 1° MKs. Panel (i) shows averagedCa++ transients in 1° MK in response to UVB, their augmentation byco-exposure to ET1 peptide, and their significant attenuation by eitherGSK205, which inhibits TRPV4, or ET-convertase inhibitor CGS35066, whichblocks ET1 proteolytic processing. Panel (ii) shows ET-augmented,UVB-induced Ca++ transients as in (i), but in this case, where they areattenuated by selective antagonism of ET(R)-A (BQ123) and ET(R)-B(BQ788). Panel (iii) illustrates the complete elimination of theET1-augmented Ca++ transients when both subtypes of ET(R) are blocked.(FIG. 7B) 4α-PDD-evoked Ca++ signaling in 1° MKs—ET1-related findings.Left-hand panel shows Ca++ transients (as per fura-2 ratiometricimaging) in response to the selective TRPV4 activator 4α-PDD. Asignificant increase in the response can be observed by co-applicationof ET1, and this is partially dependent on ET(R)-A and completelydependent on ET(R)-B. (FIG. 7C) Upregulation of ET1 in mouse paw inresponse to UVB. Immunohistochemistry reveals a significantly strongerET1 signal in UVB-exposed skin of oil-vehicle-treated (TRPV4+) ratherthan tamtreated iKO mice. Quantifications are for n=3 mice/group. ***denotes statistically significant (p<0.001, t-test). (FIG. 7D)Nocifensive behavior in response to ET1 footpad injection depends onepidermal TRPV4. Bar diagram summarizes behavioral findings for Trpv4−/−vs. WT and for oil-treated vs. tam-treated iKO mice. Note that in WT andoil-treated iKO mice, footpad injection of ET1 leads to significantlevels of mechanical allodynia. Trpv4−/− and tam-treated iKO mice failto respond; n≥7 mice/group, **p<0.01, ANOVA.

FIGS. 8A-8D: Central role for KC TRPV4 in UVB-evoked Ca⁺⁺ signaling andnocifensive behavior—ET1-related supplementary findings. (FIG. 8A)Augmentation of GSK101-evoked Ca⁺⁺ signaling by ET1. Shown are averagedCa⁺⁺ measurements (fura-2) in response to 5 nM GSK101. Note the increasein signal in response to co-exposure to ET1. (FIG. 8B) ET1 secretion bynon-stimulated 1° MK depends on TRPV4 and PLC. Shown are relative ET1concentrations (determined by ELISA, pg/mL; vehicle-treated and WTcontrol normalized to 100) in supernatant of non-stimulated 1° MK. Notethe clear dependence on TRPV4, as indicated by a 50% reduction inTrpv4^(−/−) 1° MK. Moreover, there is a significant down-regulation byspecific inhibition of TRPV4, which is dose-dependent (two doses ofGSK205) and can be mediated by two different compounds (GSK205, RN1734).There is also down-regulation of ET1 secretion by a specific inhibitorof PLC (U73122), and by an ET-convertase inhibitor, CGS35066, whichserved as a control compound. In addition, PLC-inhibitor robustlyaffects ET1 secretion in WT and Trpv4^(−/−) 1° MK. (FIG. 8C) ET1expression by UVB-exposed 1° MK depends on TRPV4 andPLC—immunocytochemistry. Shown is specific ET1 immunolabeling in 1° MK,exposed to UVB using the UVB-LEDs, as for Ca⁺⁺ imaging. Use of theUVB-LED device precluded application of a ET1 ELISA, only irradiatedcells could be examined. Note the significant down-regulation of ET1immunoreactivity by specific inhibition of TRPV4 (two differentcompounds, GSK205, RN1734), by PLC inhibition (U73122), also byinhibition of ET-convertase (CGS35066). (FIG. 8D) ET1 expression byUVB-exposed 1° MK depends on TRPV4 and PLC—quantification ofimmunocytochemistry. Densitometric measurements of n≥25 cells percondition, background subtracted, are shown, indicating a significantupregulation of ET1 in response to UVB (*p<0.05 ANOVA), and significantdown-regulation vs. control-treated and UVB-exposed cells for treatmentswith selective TRPV4 antagonists (GSK205, RN1734), PLC-inhibitor U73122and ET-convertase inhibitor CGS35066; *p<0.05, t-test; #p<0.05 ANOVA.

FIGS. 9A-9H: UVB-evoked inflammasome activation in keratinocytes dependson TRPV4. (FIG. 9A) Caspase-1 immunolabeling in footpad skin in responseto UVB. Representative images are from sections of skins beforestimulation (control) or 48 hours post-UVB exposure. Bars=20 μm. (FIG.9B) Quantifications of caspase-1 immunolabeling. Bar diagrams showdensitometry, n≥3 animals/group. Comparisons: UVB exposed WT vs.Trpv4−/− and iKO+oil vs. iKO+tam. **p<0.01 ANOVA. (FIG. 9C) Westernblotting for caspase-1 from 1° MK±UVB-exposure. Note that caspase-1levels, in particular cleaved caspase-1 (lower band), are elevated inUVB-exposed WT cells, but there is a complete absence of bothprocaspase-1 and cleaved caspase-1 in 1° MK from Trpv4−/− mice. (FIG.9D) IL-1β is induced upon UVB-exposure and is dependent on TRPV4.Anti-IL-1β immunofluorescence, otherwise as in panel A. (FIG. 9E)Quantifications of IL-1β immunolabeling, n≥3 animals/group. Comparisons:UVB exposed WT vs. Trpv4−/− and iKO+oil vs. iKO+tam, **p<0.01 ANOVA.(FIG. 9F) IL-1β concentrations in interstitial fluid of UVB-exposedfootpad. IL-1β levels (ELISA) are shown in lavaged interstitial fluid.Note strong up-regulation in WT and oil-treated iKO mice after UVB, incontrast significant attenuation in Trpv4−/− and tam-treated iKO mice.n≥5 mice/group, **p<0.01 ANOVA. (FIG. 9G) CXCL5 is induced uponUVB-exposure and is dependent upon TRPV4. Anti-CXCL5 immunolabeling,otherwise as in panel A. (FIG. 9H) Quantifications of CXCL5immunolabeling, n≥3 animals/group. Comparisons: UVB exposed WT vs.Trpv4−/− and iKO+oil vs. iKO+tam, **p<0.01 ANOVA.

FIGS. 10A-10B: Epidermal TRPV4, ET1 and IL-1β are elevated inphotodermatitis as compared to healthy human skin. (FIG. 10A)Representative micrographs of TRPV4, ET1 and IL-1β distribution in theepidermis of acute photodermatitis, as compared to healthy human skin.Immunostaining for each antigen is increased in acute photodermatitisvs. healthy skin. Scale-bars=50 μm (left), 100 μm (middle), 50 μm(right). (FIG. 10B) Morphometric analysis for immunoreactive TRPV4, ET1and IL-1β. Findings reveal significantly increased immunolabeling forall three proteins in acute photodermatitis as compared to healthy humanskin (n=3 subjects for normal, healthy skin, and 3 patients for acute UVphotodermatitis).

FIG. 11 : Exemplars of human chronic photodermatitis. Upper panel showsnormal healthy human skin, as displayed in FIG. 10A, for comparison.Lower panel shows examples of chronic photodermatitis with elevatedexpression of TRPV4, in spinous and basal layers, ET1 (throughout) andIL-1β (throughout). In comparison to acute photodermatitis, note reducedinterstitial intraepidermal edema. Bar=50 μm.

FIGS. 12A-12D: External-topical application of a selective TRPV4inhibitor attenuates UVB-evoked nocifensive behavior and inflammation.(FIG. 12A) UVB-induced nocifensive behavior. Pain behavior is attenuatedby topical application of GSK205. The left-hand diagram shows withdrawalthresholds after UVB-exposure in response to noxious thermal cues(Hargreaves' test), and their modulation by two doses of topicallyapplied GSK205 (1 mM and 5 mM; applied 60′ and 10′ pre-exposure). Thehigher dose led to a significant attenuation of thermal allodynia at 48hours post-UVB; n=6 mice/group; **p<0.01 ANOVA. The righthand diagramshows development of moderate thermal allodynia in Trpv4−/− mice, andsimilar sensitization for vehicle-treated vs. 5 mM GSK205-treated mice,indicating lack of off-target effects of the compound at 5 mM; n=5mice/group. (FIG. 12B) GSK205-treatment attenuates keratinocyteexpression of IL-1β in UVB-exposed footpad—representative micrographs.Bars=20 μm. (FIG. 12C) GSK205-treatment attenuates keratinocyteexpression of IL-1β in UVB-exposed footpad quantifications. Bar diagramsshow densitometry results from n=3 mice/group, **p<0.01 ANOVA. (FIG.12D) GSK205-treatment attenuates secretion of IL-1β by UVB-exposed 1°MK. IL-1β concentrations in supernatant (ELISA), are shown in responseto UVB. Cells were cultured+/−5 μM GSK205. Note prevention of increasein IL-1β secretion in response to UVB upon treatment with GSK205.**P<0.01 ANOVA.

FIGS. 13A-13B: Topical application of a selective TRPV4 inhibitorattenuates UVB-evoked nocifensive behavior by suppressing upregulationof pro-algesic/algogenic mediators in murine keratinocytes—Findings forCXCL5 and IL6. (FIG. 13A) GSK205-treatment attenuates keratinocyteexpression of CXCL5 in UVB-exposed footpad—micrographs and quantitation.As in FIG. 12B, specific immunolabeling for CXCL5, which is selectivelyupregulated in footpad keratinocytes in response to UVB, noteattenuation with GSK205 treatment. Bar diagrams show densitometry ofCXCL5 immunolabeling, n=3 animals per group, sections analyzed peranimal. Note significant differences for vehicle-treated mice betweenUVB-exposed and non-exposed, no such difference for mice treatedtopically with 5 mM GSK205. Comparison UVBexposed between vehicle andGSK205-treated, **p<0.01 ANOVA. Bar=20 μm. (FIG. 13B) GSK205-treatmentattenuates keratinocyte expression of IL-6 in UVB-exposedfootpad—micrographs and quantification. As in FIG. 12B and FIG. 13A,specific immunolabeling for IL-6, demonstrates similar regulation ofIL-6 as for CXCL5 and IL-1β. Comparison UVB-exposed between vehicle andGSK205-treated, **p<0.01 ANOVA. Bar=20 μm.

FIGS. 14A-140 : External-topical application of a selective TRPV4inhibitor attenuates UVB-evoked nocifensive behavior and inflammation.(FIG. 14A) UVB-photodermatitis is attenuated in mice treated withGSK205. Representative H&E micrographs of paw-pad skin are shown,bars=20 μm. Treatment of UVB-exposed skin with GSK205 improved the skinarchitecture in mice as compared to vehicle-treated mice after 24 hours.(i) and (iii) Representative skin sections of UVB-inducedphotodermatitis after GSK205 treatment showed markedly reducedinflammatory infiltrate, less spongiosis and dermal-epidermal blisterswith remaining epidermal thickening. (ii) and (iv) Vehicle-treated miceafter UVB-induced photodermatitis were characterized by signs of severeacute photodermatitis such as spongiosis, epidermal hyperkeratosis,disrupted dermal-epidermal border (blister), and a marked inflammatoryinfiltrate with dilated blood vessels and dermal edema (arrows). Alsonote the erythrocyte accumulation in blood vessels indicative ofdermatitis. (FIG. 14B) Topical treatment with a TRPV4-specific inhibitorattenuates upregulation of algogenic ET1/Edn1. Edn1 mRNA was determinedby qPCR after extraction of total RNA from paw-pad epidermis. Invehicletreated skin, note increase of Edn1 expression with earlyup-regulation at the 2 hour time-point, and sustained elevation up tothe 24 hour time-point. This time-course resembles that seen in WTcontrol mice, when comparing to Trpv4−/− (FIG. 15 ). Importantly,topical treatment with 5 mM GSK205 results in complete lack of thisregulation; n=4 mice/group, *p<0.05 ANOVA. (FIG. 14C) GSK205 does notfunction as sunscreen. Schematic illustrates the experimental set-up.(FIG. 14D) GSK205 does not function as sunscreen. The bar diagram showsresults from n=7-8 mice/group, note absence of a change in UVBpermeation with 5 mM GSK205, topically applied as for (B), vs.vehiclecontrol, yet significantly reduced with sunscreen SPF100.

FIG. 15 : Upregulation of ET1/End1 in mouse paw in response to UVB. Edn1mRNA was determined by qPCR after isolation of total RNA from paw-padepidermis. Note the early increase, at the 2 hour time-point, in WTcontrol epidermis, and its complete lack in Trpv4−/−, and sustainedupregulation at 24 hours, slightly reduced vs. 2 hour time-point, againcomplete lack of upregulation in Trpv4−/−. Quantifications are for n=4mice/group. ** denotes statistically significant (p<0.01, t-test).

FIGS. 16A-16B: Skin UVB permeability testing. (FIG. 16A) Experimentalset-up for testing of skin permeability to UVB. (FIG. 16B) Results fromFIG. 16A. Note that intensity is 70% within 500 μm radius to the centerof the UV beam.

FIG. 17 : Role of TRPV4 in itch transmission in mice in vivo. Histamine(10%) was injected intracutaneously into the cheek of C57b/6 control(WT) or TRPV4 knockout mice (Trpv4 pan-knockout; n=6 per group). Over 30min, TRPV4 null mice showed a significant reduction ((p<0.01) t-test))of itch-behavior as compared to WT mice.

FIG. 18 : The role of TRPV4 in itch. Shown is a graph of scratchingbehavior after administration of a pruritogen in mice with TRPV4deletion in keratinocytes after induction of the TRPV4 knockout, ascompared to mice without induction. Without induction, the mice functionas wild-type control mice (Moore et al. Proc. Natl. Acad. Sci. U.S.A.2013, 110, E3225-E3234). Compared to control mice, scratch behavior wassignificantly reduced for mice in which TRPV4 channels had beenselectively deleted in skin keratinoctyes.

FIG. 19 : Compound 16-19. Compound 16-8/18 h was designed as a hybrid ofcompounds 16-8 and 16-18. Compound 16-19 may also be referred to hereinas 16-8/18hy.

FIG. 20 : Inhibition of calcium ion flux through TRPV4. Compounds of thepresent invention demonstrated inhibitory activity against TRPV4 andwere stronger antagonists that GSK205.

FIG. 21 : Treatment of pain. Compounds as disclosed herein attenuatednocifensive behavior in mice.

FIG. 22 : Treatment of pain after UVB exposure. Compounds as disclosedherein attenuated nocifensive behavior in a mouse model for sunburn.

FIG. 23 : Effect on TRPA1. Compounds as disclosed herein inhibitedTRPA1, as indicated by measuring calcium transience.

FIG. 24 : Effect on TRPV1, TRPV2, and TRPV3. Compounds as disclosedherein did not inhibit TRPV1, TRPV2, or TRPV3, as indicated by measuringcalcium transience.

FIG. 25 : Modifications of tool compound GSK205 for improved targetingof TRPV4. The synthesized compounds differed in the highlighted part ofthe molecule, changed residue indicated with arrow. Compound 16-19compound was synthesized to incorporate two modifications from twocompounds, 16-8 and 16-18, found most potent in anti-TRPV4 screeningassays (see FIG. 26 ).

FIGS. 26A-26B: Assessment of compounds in N2a cells with directedexpression of TRPV4. (FIG. 26A) Ca++ imaging screening of all compoundsin N2A cells with directed expression of TRPV4 (rat). The cells werestimulated with TRPV4-selective activator compound, GSK101 (5 nM) in thepresence of 5 μM of the respective inhibitor. The number on each barcorresponds to average peak ΔCa++ concentrations in ≈100 cells. Inset:micrographs of pseudo-colored cells before and after activation with 5nM GSK101, in addition note the corresponding time course of theaveraged Ca++ signal (fura-2 Ca++ imaging). Except for compound 16-430,the difference to vehicle control reach the level of statisticalsignificance p<0.01 (one-way ANOVA). (FIG. 26B) Dose-response of themost potent, “winner” compounds in TRPV4-expressing N2a cells. The IC₅₀were; 0.45±0.05 μM (16-8), 0.59±0.12 μM (16-18), 0.81±0.1 μM (16-19),4.19±0.71 μM (GSK205). Plot generated from averaged peak ΔCa++concentration of 75 cells per data-point.

FIGS. 27A-27B: TRPV4 channel inhibition by compounds 16-8 and16-19-patch-clamp e-phys. (FIG. 27A) Current-voltage relationship ofTRPV4-mediated currents after activation with 5 nM GSK101. Recordingswere performed in TRPV4-GFP+N2a cells. The representative tracesrepresent an average of ≈12 sweeps. In all experiments, cells werepreincubated with the respective compound (5 μM) for 5 minutes. (FIG.27B) Average current densities at −100 mV/+100 mV were significantlydiminished by inhibitors (*p<0.05; one-way ANOVA; n≥5 cells/group).

FIGS. 28A-28B: Compound 16-8 inhibits TRPV4 in I° cells more potentlythan GSK205. (FIG. 28A) 1° (primary) articular chondrocytes (pig);dose-response comparison between the most potent compound, 16-8, andGSK205 in response to stimulation with 5 nM GSK101. Inset: Chondrocytesresponding to activation with GSK101, fura-2 Ca++ imaging; right-handimage taken at 5 sec after GSK101 application. 16-8 was significantlymore potent than GSK205 (mean±SEM, n=6 independent expts, n≥25cells/expt; *p<0.05, t-test). Ordinate shows average peak ΔCa++concentrations. (FIG. 28B) 1° (primary) astrocytes (rat); dose-responsecomparison between 16-8 and GSK205 in response to 5 nM GSK101. Inset:Astrocytes responding to activation with GSK101; right-hand image takenat 5 sec after GSK101 application (mean±SEM, n=5 independent expts, 200cells/expt; *p<0.05, t-test). Ordinate shows average peak ΔCa++concentrations.

FIGS. 29A-29B: Compounds 16-8 and 16-19 also potently inhibit TRPA1, notTRPV1-3. (FIG. 29A) Specificity vs TRPV1-3. Both 16-8 and 16-19 (5 μMeach) compounds did not inhibit TRPV1, -2 or -3 channels (all mouseisoforms), directed over-expression in N2a cells and subsequent Ca++imaging. Mean±SEM is shown, ≥100 cells per condition. (FIG. 29B)Dose-dependent inhibition of TRPA1 (mouse, directed expression in N2acells) by GSK 205, 16-8 and 16-19, activation with 100 μM mustard oil,resulting in IC₅₀ of 5.56±0.4 μM (GSK205), 0.41±0.37 μM (16-19),0.43±0.3 μM (16-8). Plot generated from averaged peak ΔCa++concentration of 75 cells per data-point.

FIGS. 30A-30B: Cellular toxicity studies of compounds 16-8 and 16-19.N2a cells were subjected to increasing concentrations of compounds 16-8and 16-19, resulting cell viability was analyzed for the next 48 h.(FIG. 30A) Time course of cell viability in the presence of variousconcentrations of 16-8. Note clear reduction at 40 and 80 μM. (FIG. 30B)As in (FIG. 30A), for compound 16-19, with similar outcome.Representative result of 2 independent experiments.

FIGS. 31A-31D: 16-8 and 16-19 effectively attenuate formalin-evokedtrigeminal irritant pain. (FIG. 31A) Time-course of nocifensive behaviorin WT mice following whisker-pad injection of 4% formalin. The mice werepre-injected (i.p., 10 mg/kg; 15 min before formalin) with GSK205, 16-8or 16-19. Note effective reduction of nocifensive behavior in the late“neural” phase by compounds 16-8, 16-19, not by GSK205. (FIG. 31B)Cumulative response binned into 3 phases: acute phase (0-5 min),interphase (5-15 min), and late “neural” phase (15-45 min). Notesignificant reduction of nocifensive behavior in the late phase by 16-8,16-19, not GSK205 (*P<0.01 vs vehicle and GSK205, one-way ANOVA). (FIG.31C) As in (FIG. 31A), but also including Trpv4^(−/−) mice. Compoundswere applied i.p. 15 min before formalin challenge, at 10 mg/kg exceptestablished TRPA1 blocker, A967079 (25 mg/kg). Previously-establishedattenuated nocifensive behavior in early and late phase in Trpv4^(−/−)mice was recapped, which was reduced further by TRPA1 blocker, A967079.(FIG. 31D) As in (FIG. 31B), plus inclusion of Trpv4^(−/−) mice. Robusteffects of TRPA1-blocker, A967079, were mimicked equi-potently by 16-8and 16-19 for early phase, and by 16-8 for late phase, partially by16-19 for late phase. (FIG. 31A, FIG. 31C) show averaged behavioralmetrics per time-point, bars in (FIG. 31B, FIG. 31D) represent mean±SEM;for (D) *p<0.05; #P<0.005, one-way ANOVA; for all panels n=5-8mice/group.

FIGS. 32A-32F: Compound 16-8 attenuates acute pancreatitis and improvespain behavior. (FIG. 32A) Caerulein-evoked acute pancreatitis causespancreatic edema, which is eliminated by compound 16-8 (10 mg/kg,applied at 30 min before first exposure to caerulein). (FIG. 32B)Caerulein-evoked acute pancreatitis strongly elevates cellular toxicitymarker amylase in serum. Amylase is reduced, but not significantly, in16-8 treated animals. (FIG. 32C) caerulein-evoked acute pancreatitiscauses elevated myelo-peroxidase (MPO) activity in serum, a marker forinfiltration of inflammatory cells into the pancreas. MPO activity issignificantly reduced in 16-8 treated mice. (FIG. 32D) caerulein-evokedacute pancreatitis can be readily demonstrated histologically,exemplified in the micrograph panels shown. Note increased pancreasinflammation in the middle-panel vs non-inflamed pancreas invehicle-control challenged mice, and its attenuation by treatment withcompound 16-8. (FIG. 32E) Bar diagram shows quantitation of inflammatoryhistologic parameters as shown in (FIG. 32D). Note significant increaseof inflammation-index in caerulein acute pancreatitis mice, and itssignificant reduction upon treatment with compound 16-8. (FIG. 32F)Caerulein-evoked acute pancreatitis causes pain behavior, significantlyreduced by compound 16-8. Note greatly reduced activity over the 6 htest period in caerulein-induced acute pancreatitis. This nocifensivebehavior is greatly improved in response to systemic application ofcompound 16-8. Results are expressed as mean±SEM; n=6 mice/group;*P<0.05 (one-way ANOVA).

FIGS. 33A-33E: Pharmaco-kinetics/pharmaco-tox of compounds 16-8, 16-19and GSK205 in-vivo. (FIG. 33A) Concentrations of compounds 16-8, 16-19and GSK205 (10 mg/kg) in several murine tissues/organs 1 h post-i.p.injection. (FIG. 33B) Compound 16-19 time-course at 6 h and 24 h inseveral organs. 16-19 was selected because of its elevated levels at the1 h time-point, and based on the estimate that 16-19 is more lipophilicthan 16-8 and GSK205. Note that metrics at 6 h are invariably higherthan at 24 h. All values are appreciably higher than at 1 h. (FIG. 33C)Concentrations of 16-8, 16-19 and GSK205 in fat and pancreas after onehour. Note lower concentration of 16-8 vs. 16-19 and GSK205, yet aboveits IC50. (FIG. 33D) Concentrations of 16-8 in the pancreas at 1 h and24 h time-points. (FIG. 33E) Structural stability of compound 16-19 inplasma as suggested by stable concentration after 4 h/37° C. Results areexpressed as means±SEM, n=6 mice/experimental group for all experiments.

FIGS. 34A-34C: Absence of cardiac, renal and hepatic toxicty ofcompounds 16-8, 16-19. (FIG. 34A) Heart rate time-course after i.p.injection (10 mg/kg) of compounds. There was no significant differencein heart rates between vehicle and compounds. (FIG. 34B) Serum creatininwas not significantly elevated in animals treated with 16-19 and 16-8 vsvehicle control. (FIG. 34C) Serum alanin-amino-transferase (ALT) levelswere not significantly elevated in animals treated with 16-19 and 16-8vs vehicle control. n=6 mice/group for all experiments.

FIG. 35 : General synthetic scheme for compounds 16-08 to 16-19.Reagents and conditions: (i) K₂CO₃, CH₃CN. (ii) Zn, MeOH, 12M HCl. (iii)1,1′-Thiocarbonyldiimidazole. (iv) 7M NH₃ in MeOH. (v) EtOH, reflux.

DETAILED DESCRIPTION

The disclosure relates to treating a subject's skin or wounds in orderto sanitize them and remove any contaminating and possibly harmfulbacteria or other microorganisms. The disclosure also relates todiminishing pain, inflammation, and/or irritation that may be caused byiodine or a local anesthetic. Described herein are compositions andmethods for sanitizing a subject, anesthetizing a subject, and treatingand/or preventing a dermatological disorder. The skin functions as anessential barrier between the external environment and the vertebrateorganism. Keratinocytes in the skin absorb UV-light, leading to skininflammation, pain, and itch after over-exposure, which subsequentlyleads to skin pigmentation. The inventors have identified that the skin,in particular its epidermal epithelia, is substantially involved insensory transduction. A mouse model of sunburn is described herein andused to induce a state of lowered sensory thresholds evoked by alimited, self-resolving inflammation in response to UV spectrum oflight. UV-evoked lowering of sensory thresholds shares major hallmarksof pathological pain, which is a valuable feature of this model.

1. DEFINITIONS

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. In case of conflict, the present document, includingdefinitions, will control. Preferred methods and materials are describedbelow, although methods and materials similar or equivalent to thosedescribed herein can be used in practice or testing of the presentinvention. All publications, patent applications, patents and otherreferences mentioned herein are incorporated by reference in theirentirety. The materials, methods, and examples disclosed herein areillustrative only and not intended to be limiting.

The terms “comprise(s),” “include(s),” “having,” “has,” “can,”“contain(s),” and variants thereof, as used herein, are intended to beopen-ended transitional phrases, terms, or words that do not precludethe possibility of additional acts or structures. The singular forms“a,” “and” and “the” include plural references unless the contextclearly dictates otherwise. The present disclosure also contemplatesother embodiments “comprising,” “consisting of” and “consistingessentially of,” the embodiments or elements presented herein, whetherexplicitly set forth or not.

For the recitation of numeric ranges herein, each intervening numberthere between with the same degree of precision is explicitlycontemplated. For example, for the range of 6-9, the numbers 7 and 8 arecontemplated in addition to 6 and 9, and for the range 6.0-7.0, thenumber 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 areexplicitly contemplated.

Definitions of specific functional groups and chemical terms aredescribed in more detail herein. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; Carruthers, SomeModern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987; the entire contents of each of whichare incorporated herein by reference.

The term “acyl” or “carbonyl” refers to the group —C(O)R wherein R isselected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocyclyl, heteroaryl, arylalkyl, cycloalkylalkyl,heteroarylalkyl and heterocyclylalkyl, any of which may be optionallysubstituted, e.g., with one or more substituents. For example, when R isalkyl, such a group may be referred to as an alkylcarbonyl group. Theterm “acyl” or “carbonyl” includes, for example, an alkylcarbonyl,cycloalkylcarbonyl, heterocyclylcarbonyl, arylcarbonyl, orheteroarylcarbonyl substituent, any of which may be further substituted(e.g., with one or more substituents).

The term “alkyl” refers to a straight or branched hydrocarbon chain,containing the indicated number of carbon atoms. For example, C₁-C₁₂alkyl indicates that the alkyl group may have from 1 to 12 (inclusive)carbon atoms, and C₁-C₄ alkyl indicates that the alkyl group may havefrom 1 to 4 (inclusive) carbon atoms. An alkyl group may be optionallysubstituted. Examples of C₁-C₄ alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, sec-butyl and tert-butyl.

The term “alkylene” refers to divalent hydrocarbon groups having 1 to 6carbon atoms such as methylene, ethylene, propylene, and butylene.Alkylene groups include C₁-C₂ alkylene, or C₁-C₃ alkylene, or C₁-C₄alkylene.

The term “alkenyl” refers to a straight or branched hydrocarbon chainhaving one or more double bonds. Examples of alkenyl groups include, butare not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenylgroups. One of the double bond carbons may optionally be the point ofattachment of the alkenyl substituent. An alkenyl group may beoptionally substituted.

The term “alkynyl” refers to a straight or branched hydrocarbon chainhaving one or more triple bonds. Examples of alkynyl groups include, butare not limited to, ethynyl, propargyl, and 3-hexynyl. One of the triplebond carbons may optionally be the point of attachment of the alkynylsubstituent. An alkynyl group may be optionally substituted.

The term “aryl” or “aromatic” refers to an aromatic monocyclic,bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atomcapable of substitution can be substituted (e.g., with one or moresubstituents). Examples of aryl moieties include, but are not limitedto, phenyl, naphthyl, and anthracenyl. An aromatic amine is an arylgroup substituted with one or more amino groups. An aromatic alcohol isan aryl group substituted with one or more hydroxyl groups. Botharomatic amines and aromatic alcohols may be further substituted withother substitutents.

The term “arylalkyl” refers to an alkyl moiety in which an alkylhydrogen atom is replaced with an aryl group. Arylalkyl includes groupsin which more than one hydrogen atom has been replaced with an arylgroup. Examples of arylalkyl groups include benzyl, 2-phenylethyl,3-phenylpropyl, 9-fluorenyl, benzhydryl, and trityl groups.

The term “carboxyl” refers to the group —C(═O)OR, wherein R is selectedfrom the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, heterocyclyl, heteroaryl, arylalkyl, cycloalkylalkyl,heteroarylalkyl, and heterocyclylalkyl any of which may be optionallysubstituted, e.g., with one or more substituents.

The term “carbonylamino” or “amido” refers to the group —C(O)NR′R″wherein R′ and R″ are independently selected from the group consistingof hydrogen, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocyclyl,heteroaryl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, andheterocyclylalkyl, or R′ and R″, together with the nitrogen to whichthey are attached, may form a ring. The groups R′ and R″ may beoptionally substituted, e.g., with one or more substituents, or when R′and R″ together with the nitrogen to which they are attached form aring, the ring may be optionally substituted, e.g., with one or moresubstituents.

The term “cycloalkyl” as used herein refers to nonaromatic, saturated orpartially unsaturated cyclic, bicyclic, tricyclic, or polycyclichydrocarbon groups having 3 to 12 carbons (e.g., 3, 4, 5, 6, or 7 carbonatoms). Any ring atom can be substituted (e.g., with one or moresubstituents). Cycloalkyl groups can contain fused rings. Fused ringsare rings that share one or more common carbon atoms. Examples ofcycloalkyl groups include, but are not limited to, cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, cyclohexadienyl,methylcyclohexyl, adamantyl, norbornyl, and norbornenyl. The term“cycloalkenyl” refers to cyclic alkenyl groups of from 4 to 8 carbonatoms having a single cyclic ring and at least one point of internalunsaturation. Any ring atom can be substituted (e.g., with one or moresubstituents).

The term “halo” or “halogen” as used herein refers to any radical offluorine, chlorine, bromine, or iodine.

The term “haloalkyl” as used herein refers to an alkyl in which one ormore hydrogen atoms are replaced with a halogen, and includes alkylmoieties in which all hydrogens have been replaced with halogens (e.g.,perfluoroalkyl such as CF₃).

The term “heteroaryl” or “heteroaromatic” as used herein refers to anaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, saidheteroatoms independently selected from O, N, S, P, and Si (e.g., carbonatoms and 1-3, 1-6, or 1-9 heteroatoms independently selected from O, N,S, P, and Si if monocyclic, bicyclic, or tricyclic, respectively). Anyring atom can be substituted (e.g., with one or more substituents).Heteroaryl groups can contain fused rings, which are rings that shareone or more common atoms. Examples of heteroaryl groups include, but arenot limited to, radicals of pyridine, pyrimidine, pyrazine, pyridazine,pyrrole, imidazole, pyrazole, oxazole, isoxazole, furan, thiazole,isothiazole, thiophene, quinoline, isoquinoline, quinoxaline,quinazoline, cinnoline, indole, isoindole, indolizine, indazole,benzimidazole, phthalazine, pteridine, carbazole, carboline,phenanthridine, acridine, phenanthroline, phenazine, naphthyridines, andpurines.

The term “heterocyclyl” as used herein refers to a nonaromatic,saturated or partially unsaturated 3-10 membered monocyclic, 8-12membered bicyclic, or 11-14 membered tricyclic ring system having 1-3heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, S, Si,and P (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of O, N, S,Si and P if monocyclic, bicyclic, or tricyclic, respectively). Any ringatom can be substituted (e.g., with one or more substituents).Heterocyclyl groups can contain fused rings, which are rings that shareone or more common atoms. Heterocyclyl groups can includeheterocycloalkenyl groups. Examples of heterocyclyl groups include, butare not limited to, radicals of tetrahydrofuran, tetrahydrothiophene,tetrahydropyran, piperidine, piperazine, morpholine, pyrroline,pyrimidine, pyrrolidine, indoline, tetrahydropyridine, dihydropyran,thianthrene, pyran, benzopyran, xanthene, phenoxathiin, phenothiazine,furazan, lactones, lactams such as azetidinones and pyrrolidinones,sultams, sultones, and the like.

The term heteroalkyl refers to a alkyl-, a alkenyl- or a alkynyl group,wherein one or more (preferably 1, 2, or 3) carbon atoms are replaced byone or more heteroatoms, said heteroatoms selected from O, N, S, Si, andP. Heteralkyl groups include, for example, an alkyloxy group, as forexample methoxy or ethoxy, or a methoxymethyl-, nitrile-,methylcarboxyalkylester- or 2,3-dioxyethyl-group. The term heteroalkylrefers furthermore to a carboxylic acid or a group derived from acarboxylic acid as for example acyl, acyloxy, carboxyalkyl,carboxyalkylester, such as for example methylcarboxyalkylester,carboxyalkylamide, alkoxycarbonyl, or alkoxycarbonyloxy.

The term “hydroxy” or “hydroxyl” refers to an —OH radical. The term“alkoxy” refers to the group —O—R wherein R is alkyl, alkenyl, alkynyl,cycloalkyl or heterocyclyl, any of which may be optionally substituted,e.g., with one or more substituents. The term “aryloxy” refers to an—O-aryl radical. The term “haloalkoxy” refers to an —O-haloalkylradical.

The term “substituent” refers to a group “substituted” on an alkyl,alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, arylalkyl, orheteroaryl group at any atom of that group. Suitable substituentsinclude, without limitation: acyl, acylamido, acyloxy, alkoxy, alkyl,alkenyl, alkynyl, amido, amino, carboxy, cyano, ester, halo, hydroxy,imino, nitro, oxo (e.g., C═O), phosphonate, sulfinyl, sulfonyl,sulfonate, sulfonamino, sulfonamido, thioamido, thiol, thioxo (e.g.,C═S), and ureido. In embodiments, substituents on a group areindependently any one single, or any combination of the aforementionedsubstituents. In embodiments, a substituent may itself be substitutedwith any one of the above substituents.

The above substituents may be abbreviated herein, for example, theabbreviations Me, Et, and Ph represent methyl, ethyl and phenyl,respectively. A more comprehensive list of the abbreviations used byorganic chemists appears in the first issue of each volume of theJournal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations used by organic chemistsof ordinary skill in the art, are hereby incorporated by reference.

For compounds, groups and substituents thereof may be selected inaccordance with permitted valence of the atoms and the substituents,such that the selections and substitutions result in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, etc.

Where substituent groups are specified by their conventional chemicalformulae, written from left to right, they optionally encompasssubstituents resulting from writing the structure from right to left,e.g., —CH₂O— optionally also recites —OCH₂—.

In accordance with a convention used in the art, the group:

is used in structural formulas herein to depict the bond that is thepoint of attachment of the moiety or substituent to the core or backbonestructure.

The term “about” as used herein as applied to one or more values ofinterest, refers to a value that is similar to a stated reference value.In certain aspects, the term “about” refers to a range of values thatfall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value).

“Administration” or “administering” refers to delivery of a compound orcomposition by any appropriate route to achieve the desired effect.Administration may include any convenient route of administration,whether systemically/peripherally or at the site of desired action,including but not limited to, oral (e.g. by ingestion); topical(including e.g. transdermal, intranasal, ocular, buccal, andsublingual); pulmonary; respiratory (e.g. by inhalation or insufflationtherapy using, e.g. an aerosol, e.g. through mouth or nose); rectal;vaginal; parenteral, for example, by injection, including subcutaneous,intradermal, intramuscular, intravenous, intraarterial, intracardiac,intrathecal, intraspinal, intracapsular, subcapsular, intraorbital,intraperitoneal, intratracheal, subcuticular, intraarticular,subarachnoid, and intrasternal; by implant of a depot, for example,subcutaneously or intramuscularly. In certain embodiments,administration may be topical. “Co-administered” refers to simultaneousor sequential administration. A compound or composition may beadministered before, concurrently with, or after administration ofanother compound or composition. One skilled in the art can select anappropriate dosage and route of administration depending on the patient,the particular disease, disorder, or condition being treated, theduration of the treatment, concurrent therapies, etc. In certainembodiments, a dosage is selected that balances the effectiveness withthe potential side effects, considering the severity of the disease,disorder, or condition (e.g., skin inflammation, pain, or itch).

The terms “control,” “reference level,” and “reference” are used hereininterchangeably. The reference level may be a predetermined value orrange, which is employed as a benchmark against which to assess themeasured result. “Control group” as used herein refers to a group ofcontrol subjects or cells. The predetermined level may be a cutoff valuefrom a control group. The predetermined level may be an average from acontrol group. Cutoff values (or predetermined cutoff values) may bedetermined by Adaptive Index Model (AIM) methodology. Cutoff values (orpredetermined cutoff values) may be determined by a receiver operatingcurve (ROC) analysis from biological samples of the patient group. ROCanalysis, as generally known in the biological arts, is a determinationof the ability of a test to discriminate one condition from another,e.g., to determine the performance of each marker in identifying apatient having CRC. A description of ROC analysis is provided in P. J.Heagerty et al. (Biometrics 2000, 56, 337-44), the disclosure of whichis hereby incorporated by reference in its entirety. Alternatively,cutoff values may be determined by a quartile analysis of biologicalsamples of a patient group. For example, a cutoff value may bedetermined by selecting a value that corresponds to any value in the25th-75th percentile range, preferably a value that corresponds to the25th percentile, the 50th percentile or the 75th percentile, and morepreferably the 75th percentile. Such statistical analyses may beperformed using any method known in the art and can be implementedthrough any number of commercially available software packages (e.g.,from Analyse-it Software Ltd., Leeds, UK; StataCorp LP, College Station,Tex.; SAS Institute Inc., Cary, N.C.). The healthy or normal levels orranges for a target or for a protein activity may be defined inaccordance with standard practice. A control may be a subject, or asample therefrom, whose disease state is known. A control may includecells, referred to as “control cells.” The subject, or sample therefrom,may be healthy, diseased, diseased prior to treatment, diseased duringtreatment, or diseased after treatment, or a combination thereof.

“Effective amount” refers to a dosage of a compound or compositioneffective for eliciting a desired effect, commensurate with a reasonablebenefit/risk ratio. This term as used herein may also refer to an amounteffective at bringing about a desired in vivo effect in an animal,preferably, a human, such as reduction in skin inflammation, reductionin pain, or reduction in itch.

“Polynucleotide” as used herein can be single stranded or doublestranded, or can contain portions of both double stranded and singlestranded sequence. The polynucleotide can be nucleic acid, natural orsynthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where thepolynucleotide can contain combinations of deoxyribo- andribo-nucleotides, and combinations of bases including uracil, adenine,thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine,and isoguanine. Polynucleotides can be obtained by chemical synthesismethods or by recombinant methods.

A “peptide” or “polypeptide” is a linked sequence of two or more aminoacids linked by peptide bonds. The polypeptide can be natural,synthetic, or a modification or combination of natural and synthetic.Peptides and polypeptides include proteins such as binding proteins,receptors, and antibodies. The terms “polypeptide”, “protein,” and“peptide” are used interchangeably herein. “Primary structure” refers tothe amino acid sequence of a particular peptide. “Secondary structure”refers to locally ordered, three dimensional structures within apolypeptide. Secondary structure may include beta-sheet andalpha-helices. These structures are commonly known as domains, e.g.,enzymatic domains, extracellular domains, transmembrane domains, poredomains, and cytoplasmic tail domains. Domains are portions of apolypeptide that form a compact unit of the polypeptide and aretypically 15 to 350 amino acids long. Exemplary domains include domainswith enzymatic activity or ligand binding activity. Typical domains aremade up of sections of lesser organization such as stretches ofbeta-sheet and alpha-helices. “Tertiary structure” refers to thecomplete three dimensional structure of a polypeptide monomer.“Quaternary structure” refers to the three dimensional structure formedby the noncovalent association of independent tertiary units. A “motif”is a portion of a polypeptide sequence and includes at least two aminoacids. A motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids inlength. In some embodiments, a motif includes 3, 4, 5, 6, or 7sequential amino acids.

“Subject” as used herein can mean a mammal that wants or is in need ofthe herein described methods or compositions. The subject may be a humanor a non-human animal. The subject may be a mammal. The mammal may be aprimate or a non-primate. The mammal can be a primate such as a human; anon-primate such as, for example, dog, cat, horse, cow, pig, mouse, rat,camel, llama, goat, rabbit, sheep, hamster, and guinea pig; or non-humanprimate such as, for example, monkey, chimpanzee, gorilla, orangutan,and gibbon. The subject may be of any age or stage of development, suchas, for example, an adult, an adolescent, or an infant. As used herein,a “subject in need of treatment” refers to a subject in need of thecompositions and methods detailed herein. The subject may have a surfacein need of sanitizing. The subject may be in need of anesthetization.The subject may have been diagnosed with a dermatological disease ordisorder associated with skin inflammation, pain, itch, or a combinationthereof. In embodiments a subject can include human and non-humananimals. The term “non-human animals” includes all vertebrates, e.g.,non-mammals (such as chickens, amphibians, reptiles) and mammals, suchas non-human primates, domesticated and/or agriculturally useful animals(such as sheep, dogs, cats, cows, pigs, etc.), and rodents (such asmice, rats, hamsters, guinea pigs, etc.). Accordingly, embodiments ofthe methods described herein relate to treatment of a cell or tissue, acell or tissue from a subject, or a subject that may be a eukaryote, ananimal, a vertebrate animal, a mammal, a rodent (e.g., a guinea pig, ahamster, a rat, a mouse), murine (e.g., a mouse), canine (e.g., a dog),feline (e.g., a cat), equine (e.g., a horse), a primate, simian (e.g., amonkey or ape), a monkey (e.g., marmoset, baboon), an ape (e.g.,gorilla, chimpanzee, orangutan, gibbon), or a human.

“Pharmaceutically acceptable” means suitable for use in a human or othermammal. The terms “pharmaceutically acceptable carriers” and“pharmaceutically acceptable excipients” are used interchangeably andrefer to substances that are useful for the preparation of apharmaceutically acceptable composition. In certain embodiments,pharmaceutically acceptable carriers are generally compatible with theother ingredients of the composition, not deleterious to the recipient,and/or neither biologically nor otherwise undesirable. The phrases“pharmaceutically acceptable” or “pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic, or other untoward reaction when administered to an animal, orhuman. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike. In some embodiments, a carrier includes a solution at neutral pH.In some embodiments, a carrier includes a salt. In some embodiments, acarrier includes a buffered solution.

“Sample” or “test sample” as used herein can mean any sample in whichthe presence and/or level of a target is to be detected or determined,or a portion from a subject or portion of a composition as detailedherein. Samples may include liquids, solutions, emulsions, orsuspensions. Samples may include a medical sample. Samples may includeany biological fluid or tissue, such as blood, whole blood, fractions ofblood such as plasma and serum, muscle, interstitial fluid, sweat,saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid,nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid,gastric lavage, emesis, fecal matter, lung tissue, peripheral bloodmononuclear cells, total white blood cells, lymph node cells, spleencells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid,skin, or combinations thereof. In some embodiments, the sample comprisescells. In some embodiments, the sample comprises an aliquot. In otherembodiments, the sample comprises a biological fluid. Samples can beobtained by any means known in the art. The sample can be used directlyas obtained from a patient or can be pre-treated, such as by filtration,distillation, extraction, concentration, centrifugation, inactivation ofinterfering components, addition of reagents, and the like, to modifythe character of the sample in some manner as discussed herein orotherwise as is known in the art.

“Treatment” or “treating,” when referring to disease in a subject, meanssuppressing, repressing, reducing, ameliorating, or completelyeliminating the disease. Suppressing the disease involves administeringa composition to a subject after induction of the disease but before itsclinical appearance. Repressing or reducing or ameliorating the diseaseinvolves administering a composition to a subject after clinicalappearance of the disease. “Preventing” the disease involvesadministering a composition to a subject prior to onset of the disease.

2. INHIBITION OF TRPV4 AND/OR TRPA1

Provided herein are compositions and methods for inhibiting TRPV4 and/orTRPA1. Detailed herein are inhibitors of TRPV4 and/or TRPA1, as well ascompositions comprising the inhibitors. The inhibitors may be used in avariety of methods including sanitizing a surface of a subject,anesthetizing a subject, and treating various disorders and diseases.

a. TRP Channels

The compositions and methods disclosed herein relate to the discoverythat epidermal keratinocytes function prominently to orchestrateUVB-mediated inflammation and sensitization of peripheral nerve endingsin the skin. In that respect, epidermal keratinocytes have a rolesimilar to a co-sensory cell. Keratinocytes abundantly express TRPV4. Asdescribed herein, TRPV4, expressed in epidermal keratinocytes, plays arole in UV-induced inflammation and pain. The TRPV4 channel exerts itsrole as a master regulator of UVB-evoked skin inflammation andnociception through Ca++ influx into keratinocytes. This UVB-evoked,TRPV4-mediated Ca++ influx re-programs the keratinocyte to function in apro-inflammatory and pro-algesic (pro-pain) manner, via TRPV4-dependentsecretion of endothelin-1 (ET-1), which may lead to sensation of itchand skin pigmentation. TRPV4 is activated contemporaneously with UVBexposure, which leads to activation of pro-algesic pathways via secretedfactors previously demonstrated to have relevance in human pain. Asdescribed in further detail herein, mice with inducible Trpv4 deletionstargeted to keratinocytes were induced for TRPV4 deletion, subsequentlyUVB-exposed, and found to be less sensitive to noxious thermal andmechanical stimulation than control mice. Based on these studies,epidermal TRPV4 was identified as a protein involved in theorchestration of UVB-mediated skin inflammation. In mouse skin,UVB-evoked inflammasome activation and increased expression ofpro-algesic/algogenic mediators, such as IL1-β, CXCL5, ET-1, and IL-6,were TRPV4-dependent. ET-1 has been shown in humans to not only elicitpainful sensations, but to also elicit itch, when injected into theskin. Also, ET-1 has been identified as a melanogen, that is, toincrease skin pigmentation by signaling to melanocytes. In primarymurine keratinocytes, UVB caused a direct, TRPV4-dependentCa⁺⁺-response. Moreover, in mice, topical application of aTRPV4-selective inhibitor reduced UVB-evoked epidermal inflammation andpain behavior. Additionally, it was found that epidermal expression ofTRPV4, ET1, and IL-1β were increased in acute human UV-photodermatitis.The term photodermatitis is used in this application referring to skininflammation in response to UV radiation/light. This tissue response caninclude pain, irritation, itch, influx of inflammatory andpain-enhancing cells and tissue injury. The compounds as detailed hereinmay inhibit TRPV4. The compounds as detailed herein may inhibit TRPA1.The compounds as detailed herein may inhibit TRPV4 and TRPA1. Thecompounds as disclosed herein may not inhibit TRPV1, TRPV2, or TRPV3.The inhibitor may specific for TRPV4 and TRPA1.

b. TRPA1 and/or TRPV4 Inhibitor

As TRPV4 is a Ca²⁺-permeable, nonselective cation channel, someembodiments provide for a TRPA1 and/or TRPV4 inhibitor that can inhibitthe biological function of TRPA1 and/or TRPV4 (e.g., inhibit cationchannel activity, inhibit Ca++ transport and/or availability). Otherembodiments provide for a TRPA1 and/or TRPV4 inhibitor that may inhibitthe expression of mRNA encoding TRPA1 or TRPV4. Some embodiments providea TRPA1 and/or TRPV4 inhibitor that may inhibit the translation of mRNAencoding TRPA1 or TRPV4 to protein. Thus, a TRPA1 and/or TRPV4 inhibitormay indirectly or directly bind and inhibit the activity of TRPA1 and/orTRPV4 (e.g., binding activity or enzymatic activity), reduce theexpression of TRPA1 and/or TRPV4, prevent expression of TRPA1 and/orTRPV4, or inhibit the production of TRPA1 and/or TRPV4 in a cell.Inhibit or inhibiting relates to any measurable reduction or attenuationof amounts or activity, e.g., amounts or activity of TRPA1 and/or TRPV4,such as those disclosed herein. “Amounts” and “levels” of protein orexpression may be used herein interchangeably.

The inhibitors as detailed herein may inhibit TRPA1. The inhibitors asdetailed herein may inhibit TRPV4. The inhibitors as detailed herein mayinhibit TRPV4 and TRPA1. The inhibitors as disclosed herein may notinhibit TRPV1, TRPV2, or TRPV3. The inhibitor may specific for TRPV4 andTRPA1.

In some embodiments, a TRPA1 and/or TRPV4 inhibitor can increase theamount of, or the biological activity of, a protein that can reduce theactivity of TRPA1 and/or TRPV4. Inhibitors capable of increasing thelevel of such a protein may include any inhibitor capable of increasingprotein or mRNA levels or increasing the expression of the protein thatinhibits TRPV4 and/or TRPA1. In one embodiment, a TRPA1 and/or TRPV4inhibitor may comprise the protein itself. For example, a TRPA1 and/orTRPV4 inhibitor may include exogenously expressed and isolated proteincapable of being delivered to the cells. The protein may be delivered tocells by a variety of methods, including fusion to Tat or VP16 or via adelivery vehicle, such as a liposome, all of which allow delivery ofprotein-based inhibitors across the cellular membrane. Those of skill inthe art will appreciate that other delivery mechanisms for proteins maybe used. Alternatively, mRNA expression may be enhanced relative tocontrol cells by contact with a TRPA1 and/or TRPV4 inhibitor. Forexample, an inhibitor capable of increasing the level of a nativelyexpressed protein that inhibits TRPV4 and/or TRPA1 may include a geneexpression activator or de-repressor. As another example, a TRPA1 and/orTRPV4 inhibitor capable of decreasing the level of natively expressedTRPV4 and/or TRPA1 protein may include a gene expression repressor. Aninhibitor capable of increasing the level of a protein that inhibitsTRPV4 and/or TRPA1 may also include inhibitors that bind to directly orindirectly and increase the effective level of the protein, for example,by enhancing the binding or other activity of the protein. An inhibitorcapable of decreasing the level of TRPV4 and/or TRPA1 protein may alsoinclude compounds or compositions that bind to directly or indirectlyand decrease the effective level of TRPV4 and/or TRPA1 protein, forexample, by inhibiting or reducing the binding or other activity of theTRPV4 and/or TRPA1 protein.

The amount or level of expression of a biomolecule (e.g., mRNA orprotein) in a cell may be evaluated by any variety of techniques thatare known in the art. For example, the inhibition of the level ofprotein expression (e.g., TRPV4 and/or TRPA1) may be evaluated at theprotein or mRNA level using techniques including, but not limited to,Western blot, ELISA, Northern blot, real time PCR, immunofluorescence,or FACS analysis. For example, the expression level of a protein may beevaluated by immunofluorescence by visualizing cells stained with afluorescently-labeled protein-specific antibody, Western blot analysisof protein expression, and RT-PCR of protein transcripts. The expressionlevel of TRPA1 and/or TRPV4 may be compared to a control. The comparisonmay be made to the level of expression in a control cell, such as anon-disease cell or other normal cell. Alternatively the control mayinclude an average range of the level of expression from a population ofnormal cells. Alternatively, a standard value developed by analyzing theresults of a population of cells with known responses to therapies oragents may be used. Those skilled in the art will appreciate that any ofa variety of controls may be used.

A TRPA1 and/or TRPV4 inhibitor may include one or more compounds andcompositions. In some embodiments, a TRPA1 and/or TRPV4 inhibitorcomprises a compound. In some embodiments, a TRPA1 and/or TRPV4inhibitor is a compound. In some embodiments, a TRPA1 and/or TRPV4inhibitor comprises a small molecule. In some embodiments, a TRPA1and/or TRPV4 inhibitor is a small molecule. A TRPA1 and/or TRPV4inhibitor may comprise a biological molecule, including nucleic acidmolecules, such as a polynucleotide having RNAi activity against TRPA1and/or TRPV4 or a substrate thereof. In some embodiments, the nucleicacid molecules include RNAs, dsRNAs, miRNAs, siRNAs, nucleic acidaptamers, antisense nucleic acid molecules, and enzymatic nucleic acidmolecules that comprise a sequence that is sufficient to allow forbinding to an encoding nucleic acid sequence and inhibit activitythereof (i.e., are complementary to such encoding nucleic acidsequences). Suitably, an RNAi molecule comprises a sequence that iscomplementary to at least a portion of a target sequence such that theRNAi can hybridize to the target sequence under physiological orartificially defined (e.g., reaction) conditions. In some embodiments anRNAi molecule comprises a sequence that is complementary such that themolecule can hybridize to a target sequence under moderate or highstringency conditions, which are well known and can be determined by oneof skill in the art. In some embodiments an RNAi molecule has complete(100%) complementarity over its entire length to a target sequence. Avariety of RNAi molecules are known in the art, and can include chemicalmodifications, such as modifications to the sugar-phosphate backbone ornucleobase that are known in the art. The modifications may be selectedby one of skill in the art to alter activity, binding, immune response,or other properties. In some embodiments, the RNAi can comprise an siRNAhaving a length from about 18 to about 24 nucleotides, about 5 to about50 nucleotides, about 5 to about 30 nucleotides, or about 10 to about 20nucleotides.

In some embodiments, the inhibitory nucleic acid molecule can bind to atarget nucleic acid sequence under stringent binding conditions. Theterms “stringent conditions” or “stringent hybridization conditions”includes reference to conditions under which a polynucleotide willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). An example ofstringent conditions include those in which hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to65° C. is performed. Amino acid and polynucleotide identity, homologyand/or similarity can be determined using the ClustalW algorithm,MEGALIGN™ (Lasergene, Wis.). Given a target polynucleotide sequence, forexample of TRPA1 and/or TRPV4 or biological substrate thereof, aninhibitory nucleic acid molecule can be designed using motifs andtargeted to a region that is anticipated to be effective for inhibitoryactivity, such as is known in the art.

In other embodiments, a TRPA1 and/or TRPV4 inhibitor comprises anantibody that can specifically bind to a protein such as TRPA1 and/orTRPV4 or a fragment thereof. Embodiments also provide for an antibodythat inhibits TRPA1 and/or TRPV4 through specific binding to a TRPA1and/or TRPV4 substrate molecule. The antibodies can be produced by anymethod known in the art, such as by immunization with a full-lengthprotein such as TRPA1 and/or TRPV4, or fragments thereof. The antibodiescan be polyclonal or monoclonal, and/or may be recombinant antibodies.In embodiments, antibodies that are human antibodies can be prepared,for example, by immunization of transgenic animals capable of producinga human antibody (see, for example, International Patent ApplicationPublication No. WO 93/12227). Monoclonal antibodies (mAbs) can beproduced by a variety of techniques, including conventional monoclonalantibody methodology, e.g., the standard somatic cell hybridizationtechnique of Kohler and Milstein, and other techniques, e.g., viral oroncogenic transformation of B-lymphocytes. Animal systems for preparinghybridomas include mouse. Hybridoma production in the mouse is very wellestablished, and immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are well known in the art. Fusionpartners (e.g., murine myeloma cells) and fusion procedures are alsoknown.

Any suitable methods can be used to evaluate a candidate active compoundor composition for inhibitory activity toward TRPA1 and/or TRPV4. Suchmethods can include, for example, in vitro assays, in vitro cell-basedassays, ex vivo assays, and in vivo methods. The methods can evaluatebinding activity, or an activity downstream of the enzyme of interest.Ex vivo assays may involve treatment of cells with an inhibitor of theinvention, followed by detection of changes in transcription levels ofcertain genes, such as TRPA1 and/or TRPV4 through collection of cellularRNA, conversion to cDNA, and quantification by quantitative real timepolymerase chain reaction (RT-QPCR). Additionally, the cell viability orinflammation may be determined after treatment with an inhibitor.

i. Compounds of Formula I

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is a compoundaccording to Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups;

D is C₁-C₃ alkylene;

E is a bond, or C₁-C₂ alkylene; and

R is selected from the group consisting of hydrogen, hydroxyl, amino,alkyl, alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring. Insome embodiments, B and C are independently a phenyl group. In someembodiments, A is phenyl or heteroaryl. In some embodiments, A ispyridnyl. In some embodiments, R is C1-C4 alkyl. In some embodiments, Ais heteroaryl, B and C are phenyl, D is ethylene, E is methylene, and Ris methyl. In some embodiments, R is ethyl.

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor excludes thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor is thefollowing compound:

In certain embodiments, the TRPA1 and/or TRPV4 inhibitor comprises thefollowing compound:

ii. Synthesis of Compounds

Compounds of Formula I may be synthesized by a variety of means known inthe art. An exemplary synthesis is detailed in the Examples and FIG. 35.

General Procedure for the S_(N)2 Displacement of 4-NitrophenethylBromide:

Powdered, oven-dried K₂CO₃ (1.5 eq.) and the amine (1.5 eq.) may besequentially to a room temperature solution of the bromide (0.33 M) inanhydrous CH₃CN. The reaction mixture may be heated to 80° C. (oil bathtemp) until analysis of the reaction mixture by LCMS indicated completeconsumption of the bromide (˜6-18 h). The mixture may be cooled to roomtemperature and diluted with brine (two volume equivalents). Theresulting emulsion may be extracted with EtOAc (2× one volumeequivalent). The combined extracts may be added to silica gel (mass ofsilica gel=2× mass of starting bromide) and the mixture may beconcentrated to dryness under reduced pressure. Flash columnchromatography (RediSepRf SiO₂, 100% CH₂Cl₂→5% MeOH in CH₂Cl₂) mayconfirm the product as a brown to amber oil.

General Procedure for the Nitro to Aniline Reduction:

A solution of the nitro compound (0.5 M in MeOH) may be cooled in anice-NaCl bath. Zinc dust (4.5 eq.) may be added in one portion followedby drop wise addition of 12M HCl (4.5 eq.) over 2-3 minutes. After 1 h,the cooling bath may be removed and the reaction mixture may be allowedto stir over night at room temperature. The following morning, themixture may be cooled in an ice-NaCl bath once again and 30% aqueousNaOH may be added drop wise until pH 14 (universal indicating pH paper)was reached. The mixture may be diluted with CH₂Cl₂ (five volumeequivalents) and stirred for 5 minutes. After this time, insolubles maybe removed at the vacuum and the filter cake may be washed with CH₂Cl₂(2×25 mL). The organic phase of the filtrate may be separated, washedwith brine (100 mL) and dried (MgSO₄).

The drying agent may be removed by filtration. Silica gel (˜5 g) may beadded and the filtrate may be concentrated to dryness under reducedpressure. Flash column chromatography (RediSepRf SiO₂, 100% CH₂Cl₂→5%MeOH in CH₂Cl₂) may confirm the product as a clear, amber oil.

General Procedure for Thiourea Formation:

A solution of the aniline (0.22 M) in anhydrous CH₂Cl₂ may be s addeddrop wise over 2-5 minutes to an ice-NaCl bath cooled solution of1,1-thiocarbonyldiimidazole (2 eq., 0.15 M) in anhydrous CH₂Cl₂. After15 minutes, the cooling bath may be removed and the reaction mixture maybe stirred at room temperature until analysis by TLC (5% MeOH in CH₂Cl₂)indicates complete consumption of the starting aniline. The mixture maybe cooled once again in an ice bath and 7M NH₃ in MeOH (10.5 eq.) may beadded drop wise over 2-5 minutes. The bath may be removed and themixture may be stirred over night at room temperature. Silica gel (massof silica gel=2× mass of starting aniline) may be added and the mixturewas concentrated to dryness under reduce pressure. Flash columnchromatography (RediSepRf SiO₂, 100% CH₂Cl₂→10% MeOH in CH₂Cl₂) mayconfirm the pure thiourea.

General Procedure for Thiazole Formation:

A mixture of the thiourea (0.1 M) in EtOH and the α-bromoacetophenonederivative (1.1 eq.) may be heated to 75° C. (oil bath temperature)until analysis by TLC (5% MeOH in CH₂Cl₂) indicates complete consumptionof the thiourea. Silica gel (mass of silica gel=2× mass of startingthiourea) may be added and the mixture may be concentrated to drynessunder reduce pressure. Flash column chromatography (RediSepRf SiO₂, 100%CH₂Cl₂→10% MeOH in CH₂Cl₂) may confirm the pure thiazole hydrobromide.

c. Compositions

In other aspects, the disclosure provides compositions comprising one ormore TRPA1 and/or TRPV4 inhibitor. The compositions may be used to treata subject's skin or wounds in order to sanitize them, or to remove orreduce contaminating and possibly harmful bacteria or othermicroorganisms. While doing so, the compositions may also reduce pain,inflammation, irritation, or a combination thereof, that may be causedby or result from a local anesthetic.

In some embodiments, the composition comprises an inhibitor and iodine.Iodine may be present in the composition as elemental iodine, or as aniodine salt, or a combination thereof. Iodine salts may include, forexample, potassium iodide and sodium iodide.

In some embodiments, the composition comprises an inhibitor and ananesthetic. In some embodiments, the anesthetic is a local anesthetic.In some embodiments, the anesthetic is not a general anesthetic. In someembodiments, the anesthetic numbs the skin of the subject. Thecomposition comprising an inhibitor and an anesthetic may be used toefficiently clean a subject's skin. The composition comprising aninhibitor and an anesthetic may be applied to the subject prior to amedical procedure, surgery, or an incision. The composition comprisingan inhibitor and an anesthetic may be applied to the subject's wound topromote healing.

Local anesthetics reduce or eliminate the tactile and pain sensationsblocking transmission in pain fibers and allow a medical professional tomanipulate anesthetized tissue without fear of causing the subject pain.Unlike general anesthesia, local anesthesia allows the subject to beawake and aware during the procedure, thus promoting subject confidenceand well-being during the medical procedure. Additionally, localanesthesia avoids the some of the risks associated with generalanesthesia, such as nausea, vomiting, and malignant hyperthermia. Manycompounds are available for use as local anesthetics. Local anestheticsmay be divided into two broad categories based on their chemicalstructure. Amide-containing anesthetics include, for example, articaine,bupivacaine, cinchocaine, etidocaine, levobupivacaine, lidocaine,mepivacaine, prilocaine, ropivacaine, and trimecaine. Another class oflocal anesthetics contains an ester chemical residue and includes, forexample, benzocaine, chloroprocaine, cocaine, cyclomethycaine,dimethocaine, piperocaine, propoxycaine, procaine (novocaine),proparacaine, and tetracaine.

The composition may comprise the TRPA1 and/or TRPV4 inhibitor incombination with a carrier, vehicle, or diluent. Embodiments provide forpharmaceutically acceptable carriers including, but not limited to,substances useful for topical, intrathecal, ocular, parenteral,intravenous, intraperitoneal intramuscular, sublingual, nasal, and oraladministration. Administration may be systemic. “Pharmaceuticallyacceptable carrier” also includes agents for preparation of aqueousdispersions and sterile powders for injection or dispersions. Examplesof pharmaceutically acceptable carriers and excipients are discussed,e.g., in Remington Pharmaceutical Science, 16th Ed. Certain exemplarytechniques and compositions for making dosage forms are described in thefollowing references: Modern Pharmaceutics, Chapters 9 and 10, Banker &Rhodes, eds. (1979); Lieberman et al., Pharmaceutical Dosage Forms:Tablets (1981); and Ansel, Introduction to Pharmaceutical Dosage Forms,2nd Ed., (1976). The carrier, vehicle, or diluent may be suitable fortopical application. In some embodiments, the carrier comprises water.In some embodiments, the carrier comprises an alcohol such as methanolor ethanol.

In certain embodiments, compositions are formulated for injection. Incertain embodiments, compositions are formulated for topicaladministration. For compositions suitable for topical administration,the composition may be combined with one or more carriers and used inthe form of cosmetic formulations. Formulations may include a foam,cream, gel, lotion, ointment, or solution. For example, a TRPA1 and/orTRPV4 inhibitor may be suitably dissolved in the alcohol of skindisinfectant gel or in lotions, creams, or other formulations. Incertain embodiments, a TRPA1 and/or TRPV4 inhibitor may be included inor added to a cosmetic formulation. In certain embodiments, a TRPA1and/or TRPV4 inhibitor may be included in or added to sun protectiontopical formulations.

For oral therapeutic administration, the composition may be combinedwith one or more carriers and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,chewing gums, foods, and the like. The percentage of the compositionsand preparations may, of course, be varied and may conveniently bebetween about 0.1 to about 100% of the weight of a given unit dosageform. The tablets, troches, pills, capsules, and the like may alsocontain the following: binders such as gum tragacanth, acacia, cornstarch or gelatin; excipients such as dicalcium phosphate; adisintegrating agent such as corn starch, potato starch, alginic acidand the like; a lubricant such as magnesium stearate; and a sweeteningagent such as sucrose, fructose, lactose or aspartame or a flavoringagent such as peppermint, oil of wintergreen, or cherry flavoring. Theabove listing is merely representative and one skilled in the art couldenvision other binders, excipients, sweetening agents and the like. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like.

The amount of a TRPA1 and/or TRPV4 inhibitor in such therapeuticallyuseful compositions is such that an effective dosage level will beobtained. The selected dosage level will depend upon a variety offactors including the activity of the particular compound employed, theroute of administration, the time of administration, the rate ofexcretion or metabolism of the particular compound being employed, theduration of the treatment, other drugs, compounds and/or materials usedin combination with the particular compound employed, the age, sex,weight, condition, general health and prior medical history of thepatient being treated, and like factors well known in the medical arts.

In general, the daily dose contains from about 0.1 mg to about 2000 mgof the active ingredient, or about 0.5 to about 60 mg of the activeingredient. This dosage form permits the full daily dosage to beadministered in one or two oral doses. More than once daily or twicedaily administrations, e.g., 3, 4, 5, or 6 administrations per day, arealso contemplated herein.

In some embodiments, as noted above, administering relates to providingan amount effective at bringing about a desired in vivo effect such asinhibition of TRPA1 and/or TRPV4 in an animal, such as a human.

d. Mouse Model

In other aspects, the disclosure provides a transgenic mouse whosegenome comprises deletions of the Trpv4 gene in keratinocytes of theepidermis. The transgenic mouse may be a knockout for the Trpv4 gene inkeratinocytes of the epidermis following keratinocyte-specificactivation and expression of a site-specific recombination enzyme.Knockout of the Trpv4 gene may be carried out by any suitable meansknown in the art. For example, the transgenic mouse may be generated byKeratin-14 promoter expression of a site-specific recombination enzyme.Site-specific recombination enzymes may include CRE recombinase. Thesite-specific recombination enzyme may be fused to a mutated estrogenreceptor. An anti-estrogen may have increased affinity to the mutatedestrogen receptor relative to wild-type estrogen. The anti-estrogen maycomprise tamoxifen. In some embodiments, addition of the anti-estrogento the transgenic mouse drives the site-specific recombination enzyme tothe nucleus and results in knockdown of expression of the Trpv4 gene. Assuch, the keratinocyte-specific deletion of the Trpv4 gene may beinduced by applying an anti-estrogen. In some embodiments, deletion ofthe Trpv4 gene can be specifically and conditionally induced inkeratinocytes of the epidermis. In some embodiments, deletion of theTrpv4 gene may be achieved by expression of a constitutively active orinducible recombination enzyme in keratinocytes of the epidermis. Insome embodiments, the transgenic mouse may exhibit reduced expressionrelative to a control of at least one of IL6, ET1, caspase1, IL1β, andCXCL5, or a combination thereof, in response to UVB exposure.

e. Methods

i. Methods of Sanitizing a Subject

Provided herein are methods of sanitizing a subject, or a surface orportion of the subject. The methods may include contacting the subjectwith a composition comprising an inhibitor and iodine, wherein theinhibitor inhibits TRPV4, TRPA1, or a combination thereof. In someembodiments, the inhibitor does not inhibit TRPV1, TRPV2, or TRPV3. Insome embodiments, the inhibitor comprises a compound according toFormula I as detailed herein. In some embodiments, the subject iscontacted with the composition for a period of time sufficient to causea reduction in the population of microorganisms on the surface. Thesurface of the subject, or a portion thereof, contacted with thecomposition may be selected from the group consisting of a skin area, awound, sore, and ulcer. In some embodiments, the composition is appliedto a medical dressing or bandage, which is then applied to the subject.

In some embodiments, the composition is administered to the subject totreat the subject's skin or wounds in order to sanitize them and reduceor remove any contaminating and possibly harmful bacteria or othermicroorganisms. In some embodiments, the composition disinfects thesubject or a surface thereof. In some embodiments, the compositionsterilizes the subject or surface thereof. In some embodiments, thecomposition has antibacterial activity. In some embodiments, thecomposition promotes healing of the skin, wound, sore, or ulcer. In someembodiments, the composition also diminishes pain, inflammation,irritation, or a combination thereof, that may be caused by or resultfrom the iodine. In some embodiments, the inhibitor reduces orattenuates pain induced by the iodine. In some embodiments, thecomposition provides local inhibition of pain. In some embodiments, thecomposition is applied to the subject prior to a medical procedure suchas surgery or an incision. In such embodiments, the composition maysanitize an area of the subject, prevent a microbial infection, reduce amicrobial infection, treat a microbial infection, or a combinationthereof. The composition may be administered prior to, during, or aftersurgery or other medical procedure.

ii. Methods of Anesthetizing a Subject

Provided herein is a method of anesthetizing a subject. The method mayinclude administering to the subject a composition comprising aninhibitor and an anesthetic, wherein the inhibitor inhibits TRPV4,TRPA1, or a combination thereof. The methods may includeco-administering an anesthetic and an inhibitor to the subject, whereinthe inhibitor inhibits TRPV4, TRPA1, or a combination thereof. In someembodiments, the inhibitor does not inhibit TRPV1, TRPV2, or TRPV3. Insome embodiments, the inhibitor comprises a compound according toFormula I as detailed herein. In some embodiments, the anesthetic is alocal anesthetic. In some embodiments, the composition diminishes pain,inflammation, irritation, or a combination thereof, that may be causedby or result from the local anesthetic. In some embodiments, thecomposition provides local inhibition of pain. In some embodiments, theinhibitor reduces or attenuates pain and/or burning induced by theanesthetic. In some embodiments, the subject has acute pulpitis or aninflamed “hot” tooth. In some embodiments, the subject has a purulentinfection of the skin or soft tissue. In some embodiments, thecomposition is administered to the acute pulpitis, inflamed “hot” tooth,or purulent infection of the skin or soft tissue of the subject. In someembodiments, the composition is applied to the subject prior to amedical procedure such as surgery or an incision. The composition may beadministered prior to, during, or after surgery, oral surgery, or otherdental or medical procedure.

iii. Methods of Treating a Disease or Disorder

The compositions and methods may be used to treat a variety of diseasesand disorders, including dermatological disorders. Provided herein aremethods of treating a disease or disorder. The methods may includeadministering to the subject an effective amount of a TRPV4 and/or TRPA1inhibitor as detailed herein.

In some embodiments, the disease or disorder that may be treated by thecompositions and methods disclosed herein may include pancreatitis (forexample, according to a model of pancreatitis induced by caerulein),epilepsy, arthritis, osteoarthritis, multiple sclerosis, stroke, CNSautoimmune conditions, traumatic brain/spinal cord injury, brain edema,CNS infections, neuro-psychiatric disorders, skeletaldegenerative-inflammatory disorders, trigeminal pain such as headaches,colitis, and sclerosis.

In other aspects, the disclosure provides methods of preventingdermatological diseases or disorders such as irritation, pain, itch,pruritus, autoimmune diseases, skin cancer (including melanoma, forexample, with topical treatment of TRPA1 and/or TRPV4 inhibitor-based UVprotection), autoimmune diseases, fibrotic disorders, pigmentationdisorders, and others as described above. In some embodiments, thedisclosure provides methods of preventing the development and/orexacerbation of Xeroderma pigmentosum, Cockayne syndrome, Bloomsyndrome, Rothmund-Thomson syndrome, and Hartnup syndrome.

The dermatological disorder may be associated with the TRPA1 or TRPV4pathway. Dermatological disorders include, but are not limited to,photo-induced inflammation, pain in diseases involving skin pain, itch,cancer, autoimmune diseases, fibrotic diseases, other acneiform orinflammatory skin diseases, and pigmentation disorders. For example,dermatological disorders may include, but are not limited to, sunburn;photoallergic reaction; phototoxic reaction; phytophotodermatitis(Berloque dermatitis); acute and chronic actinic dermatitis; atopicdermatitis exacerbation; all subtypes of rosacea includingtrigeminal-pain associated rosacea; all lupus erythematosus subtypes(systemic, discoid, subacute); atopic dermatitis; actinic prurigo;prurigo nodularis; prurigo subacuta; prurigo pigmentosa; Lichen simplex(also called neurodermatitis); diabetic pruritus; uremic pruritus;pruritus induced by metabolic (liver) diseases; pruritus induced bymalignancies like lymphoma; pruritus induced by polycythemia vera;pruritus induced by scabies; pruritus induced by bullous pemphigoid;pruritus induced by urticaria (especially but not exclusively actinicurticaria); pruritus induced by insect/arachnoid vector bite; pruritusinduced by parasitosis; melanoma; non-melanoma skin cancer (BCC, SCC);actinic keratosis and other premalignant skin cancers; mycosisfungoides; Sézary syndrome; Xeroderma pigmentosum; Cockayne syndrome;all lupus erythematosus subtypes (systemic, discoid, subacute);dermatomyositis; erythema multiforme; lichen planus; fibrotic diseasesinduced by UV-exposure (Rhinophyma, chronic actinic dermatitis, actinicreticuloid, photoaging, hyalinosis cutis et mucosae; polymorph lighteruption; Acne aestivalis; all porphyria subforms with implications onphoto-induced skin changes (erythropoetic porphyria, erythropoeticprotoporphyria, Porphyria variegate); photo-induced Herpes simplexinfection (Herpes labialis); morbus Darier; disseminated superficialactinic porokeratosis; pityriasis rubra pilaris; Bloom syndrome;Rothmund-Thomson syndrome; Hartnup syndrome photoaging; wrinkles;photo-induced inflammation; pigmentation; and pigmentation disorders.

In some embodiments, the disclosure provides a method of treating asubject wherein the method comprises administering an inhibitor of TRPA1and/or TRPV4 in a pharmaceutically acceptable composition.

iv. Methods of Reducing Skin Inflammation

In an aspect, the disclosure provides methods of reducing skininflammation in a subject in need thereof. The methods may compriseadministering to the subject an effective amount of a TRPA1 and/or TRPV4inhibitor. The skin inflammation may be related to UVB exposure.

Skin inflammation may be associated with conditions including, but notlimited to, sunburn (acute photodermatitis), photoallergic reaction,phototoxic reaction, phytophotodermatitis (Berloque dermatitis), acuteand chronic actinic dermatitis, atopic dermatitis exacerbation, androsacea.

v. Pain Management

In other aspects, the disclosure provides methods of pain management.The methods may comprise administering to at least a portion of the skinof a subject in need thereof an effective amount of a TRPA1 and/or TRPV4inhibitor. The pain may be related to UVB exposure.

Pain may be chronic or acute. Pain may be associated with or result fromconditions including, but not limited to, all subtypes of rosaceaincluding trigeminal-pain associated rosacea, reflex sympatheticdystrophy (RSD), and all lupus erythematosus subtypes (systemic,discoid, subacute).

vi. Methods of Reducing Itch

In other aspects, the disclosure provides methods of reducing itch in asubject in need thereof. ET-1 has been shown to elicit itch, and asshown in the Examples, increased expression of ET-1 was TRPV4-dependent.The methods may comprise administering to the subject an effectiveamount of a TRPA1 and/or TRPV4 inhibitor.

Itch may be chronic or acute. Itch may be associated with or result fromconditions including, but not limited to, rosacea, atopic dermatitis,actinic prurigo, prurigo nodularis, prurigo subacuta, prurigopigmentosa, Lichen simplex (also called neurodermatitis), diabeticpruritus, and uremic pruritus. Itch or pruritus may be associated withor result from conditions including metabolic (liver) diseases,malignancies like lymphoma, polycythemia vera, scabies, bullouspemphigoid, urticaria (especially but not exclusively actinicurticaria), insect/arachnoid vector bite, and parasitosis.

vii. Method of Treating Cancer

In other aspects, the disclosure provides methods of treating cancer ina subject in need thereof. The methods may comprise administering to thesubject an effective amount of a TRPA1 and/or TRPV4 inhibitor.

The cancer and related conditions may include, but are not limited to,melanoma, non-melanoma skin cancer (BCC, SCC), actinic keratosis andother premalignant skin cancers, mycosis fungoides, Sézary syndrome, andXeroderma pigmentosum.

viii. Methods of Treating an Autoimmune Disease

In other aspects, the disclosure provides methods of treating anautoimmune disease in a subject in need thereof. The methods maycomprise administering to the subject an effective amount of a TRPA1and/or TRPV4 inhibitor.

Autoimmune diseases may include, but are not limited to, all lupuserythematosus subtypes (systemic, discoid, subacute), dermatomyositis,erythema multiforme, and lichen planus.

ix. Methods of Treating a Fibrotic Disease

In other aspects, the disclosure provides methods of treating a fibroticdisease in a subject in need thereof. The methods may compriseadministering to the subject an effective amount of a TRPA1 and/or TRPV4inhibitor.

Fibrotic diseases may include conditions induced by UV-exposure, suchas, for example, Rhinophyma, chronic actinic dermatitis, actinicreticuloid, photoaging, and hyalinosis cutis et mucosae.

x. Methods of Treating Other Acneiform or Inflammatory Skin Disease

In other aspects, the disclosure provides methods of treating otheracneiform or inflammatory skin disease in a subject in need thereof. Themethods may comprise administering to the subject an effective amount ofa TRPA1 and/or TRPV4 inhibitor.

Acneiform or inflammatory skin diseases may include, but are not limitedto, polymorph light eruption, Acne aestivalis, photo-induced Herpessimplex infection (Herpes labialis), morbus Darier, disseminatedsuperficial actinic porokeratosis, Pityriasis rubra pilaris, and allporphyria subforms with implications on photo-induced skin changes suchas, for example, erythropoetic porphyria, erythropoetic protoporphyria,and Porphyria variegate.

xi. Methods of Reducing Skin Pigmentation

In other aspects, the disclosure provides methods of reducing skinpigmentation in a subject in need thereof. ET-1 has been shown to signalto skin melanocytes and function as a major melanogen (=enhancing skinpigmentation), and as shown in the Examples, increased expression ofET-1 was TRPV4-dependent. The methods may comprise administering to thesubject an effective amount of a TRPA1 and/or TRPV4 inhibitor.

Skin inflammation, pain, itch, and/or pigmentation may also beassociated with disorders including, but not limited to, Cockaynesyndrome, non-UV skin burn less than 3^(rd) degree, disturbed woundhealing, exposure and pathological response to poison ivy, and pain ofbone fractures directly adjacent to the skin such as fractures of thetibia, digits, or skull. For example, one or more of these disorders maybe symptomatic of reflex sympathetic dystrophy (RSD).

xii. Methods of Treating or Preventing Cosmetic Conditions

In other aspects, the disclosure provides methods of treating orpreventing cosmetic conditions. For example, the disclosure providesmethods of treating or preventing photoaging, wrinkles, photo-inducedinflammation, pigmentation, and pigmentation disorders. The methods maycomprise administering to the subject an effective amount of a TRPA1and/or TRPV4 inhibitor.

xiii. Methods for Identifying a Selective Inhibitor of TRPA1 and/orTRPV4

In a further aspect, the disclosure provides methods for identifying aselective inhibitor of TRPA1 and/or TRPV4. The methods may include (a)contacting a mouse with a test compound; (b) determining a biologicalactivity of TRPA1 and/or TRPV4 after contacting with the test compound;and (c) determining a control level of biological activity of TRPA1and/or TRPV4 in the absence of the test compound; (d) comparing thebiological activity of TRPA1 and/or TRPV4 from step (b) with thebiological activity of TRPA1 and/or TRPV4 from a model of TRPV4deletion, wherein the model of TRPV4 deletion includes the transgenicmouse as disclosed herein or a pan-null Trpv4−/− mouse; and (e)identifying the test compound as a selective inhibitor of TRPA1 and/orTRPV4 when at least one of (i) the TRPA1 and/or TRPV4 biologicalactivity is lower in the presence of the test compound than the TRPA1and/or TRPV4 biological activity in the absence of the test compound;(ii) the TRPA1 and/or TRPV4 biological activity in the presence of thetest compound is about the same as, or lower than, the TRPA1 and/orTRPV4 biological activity in the model of TRPV4 deletion; or (iii) anycombination of (i) and (ii).

3. EXAMPLES Example 1: Materials and Methods

Animals. The Trpv4 genomic locus was engineered so that loxP sitessurrounded exon13 which encodes TM5-6. This mutation was propagated inmice which were crossed to K14-CRE-ERtam mice, so that((Trpv4lox/lox)×(K14-CRE-ERtam))-mice could be induced by tamoxifenadministration via oral gavage for 5 consecutive days at 6 mg/day in 0.3mL cornoil, at age 2-4 months of age, plus a one-time booster two weeksafter the last application. Male and female mice were induced equally.Efficiency of knockdown was verified by qRT-PCR for Trpv4 using primerssense 5′-CCTGCTGGTCACCTACATCA (SEQ ID NO: 1) and antisense5′-CTCAGGAACACAGGGAAGGA (SEQ ID NO: 2), with the former primer locatedin exon 13. All animal experimentation described here was conducted infull compliance with NIH and Duke University internal guidelines, andunder a valid IACUC protocol.

Using the same genomic clone that was used for generating the Trpv4−/−pan-null mouse, the Trpv4 targeting construct was electroporated intomouse ES cells (C57BL6 background), and orthotopic integration wasverified by PCR and Southern blot. The engineered mutation wasintroduced into the germline by mating of chimeric mice with C57BL6 WTmice. The selection marker was deleted by FLPemediated excision of thefrt-pGK-neo-frt cassette in FLPe deleter mice. Genotyping wasaccomplished by PCR and subsequent Pacl digest (FIG. 1B).

8-12 week old mice were used throughout the experiments. Trpv4−/− mice³have been outcrossed to WT (C57BL/6J) background and genotyped byPCR^(3,15). Animals were housed in climate-controlled rooms on a 12/12 hlight/dark cycle with water and standardized rodent diet that wasavailable ad libitum. All experiments were conducted in compliance andaccordance with the guidelines of the NIH and the Institutional Animals'Care and Use Committee (IACUC) of Duke University, and under a validIACUC protocol of the Duke University IACUC. All animal methodsdescribed in this publication were approved by the Duke UniversityIACUC.

Behavioral Assessment of Withdrawal Thresholds.

Behavioral tests were performed to evaluate the decrease in withdrawalthresholds in response to mechanical von Frey hair or thermal stimuliapplied to hind paws. Tests were conducted. These withdrawal thresholdswere ascertained before and after UV exposure. Mice were exposed using aBio Rad Gel Doc 2000 UV transilluminator (302 nm) for 5 minutes with anexposure of 600 mJ/cm², 3-5 days after the last application oftamoxifen/oil.

Paw Interstitial Fluid Analysis.

48 hours after UV exposure, each animal received an intraplantarinjection of 10 μL PBS directly posteuthanasia. The interstitial fluidwas immediately collected and analyzed by ELISA (Biorad) for presence ofIL-111.

In-Vivo Topical Interventions.

ET1 injections: After determining base-line withdrawal thresholds, eachanimal received an intraplantar injection of 10 μL 100 nM ET-1 pluscontralateral vehicle. Thresholds were again evaluated 1 hour afterinjection.

GSK205 topical treatment: A viscous solution of 68% EtOH/5% glycerolplus 1 mM or 5 mM GSK205 (none for control) was applied to hindpaws byrubbing in 20 μL, applied at time-points 1 hour and again 10 min beforeUV exposure.

Formalin-induced nocifensive behavior: 4% formalin was injected into theright hindpaw. Mice were then videotaped for 50 mins and behavioranalyzed by blinded observers.

Mouse Tissue Processing for 1 μm Semithin Sections and EM.

Samples were processed and subjected to EM.

Mouse and Human Tissue Processing for Immunohistochemistry.

Routine procedures were followed, and human tissue was processed underinstitutional review-board approval (UCSF).

Primary Mouse Keratinocyte Cell Culture.

Primary mouse keratinocytes were derived from back skin of newborn mice.

Analysis of IL-1β Secreted by Cultured Keratinocytes after UV Exposure.

Before UV irradiation, culture media was replaced with PBS. The cellswere then irradiated at a dose of 50 mJ/cm² with UVB. 24 hours later,supernatants were assayed using IL-1β ELISA (R&D Systems, Minneapolis,Minn.).

Ca++ Imaging of Cultured Keratinocytes.

Ca++ imaging of 1° MK was conducted following routine procedures. ForUVB stimulation, a customized device was built. The system comprised aprinted circuit board for electrical interconnects and mechanicalsupport and an ultraviolet light-emitting diode (UV LED). Customizedprovisions at the cellular end included a quartz coverslip as the bottomof the cell culture dish plus a thermal equilibration stage (HW-101Dagan Corporation), fitted to an Olympus BX61 upright microscope. The UVLED was a III-nitride-based type (UVTOP-295 BL; Sensor ElectronicTechnology).

The operating wavelength was 295 nm (FIG. 2A), with a full-widthhalf-max of 12 nm. The optical power was 500 mW. The focal point wasaimed at the plane of the upper surface of the quartz coverslip, whichwas used to minimize UV absorption along the optical path towards thecells (FIG. 2A-C). The optical intensity at the focal point wasestimated to be 150 mW/mm².

Keratinocyte UV Irradiation Using 295 nm LED and Immunocytochemistry.

1° MK were exposed to UVB using the UV optical system (295 nm LED). 24hours later the cells were fixed and fluorescently immunolabeled forET1. Digital images were captured and subjected to morphometry.

Statistical Analysis.

Data are expressed as mean±SEM. Numeric signals or values were averagedfor their respective groups, and the statistical mean+/−standard errorof the mean were compared between groups by using a fixed-effect one-wayANOVA and post-hoc Scheffe test, or Student's t-test, or two-tailt-tests, or one-way ANOVA followed by Tukey post-hoc test at asignificance level of p<0.05.

Chemicals/Biological.

The following biologicals and compounds were used: Endothelin1; BQ123and BQ788 (ET(R) blockers for ET(R)-A and ET(R)-B; Sigma, St. Louis,Mo.), U73122 (PLC inhibitor; Tocris, Ellisville, Mo.), 4α-phorbol 12,13didecanoate (4α-PDD; TRPV4 activator; Tocris), GSK205 (TRPV4 inhibitor(Li et al., 2011; Phan et al., 2009; Vincent and Duncton, 2011)),RN-1734 (TRPV4 inhibitor; Tocris), CGS35066 (endothelin-convertingenzyme inhibitor, Tocris), isopentenyl pyrophosphate, IPP (TRPV3inhibitor; Sigma); and Camphor (TRPV3 activator; Whole Foods).

Behavioral Assessment of Withdrawal Thresholds and Nocifensive Behavior.

Behavioral tests were performed to evaluate the decrease in withdrawalthresholds in response to mechanical or thermal stimuli applied to hindpaws. These withdrawal thresholds were ascertained before and after UVBexposure. Mice were confined by plexi-glass enclosures on top of 25×26cm Bio Rad Gel Doc 2000 UV transilluminator (302 nm), and otherwiseallowed to openly explore this environment. UV-exposure lasted for 5minutes with an exposure of 600 mJ/cm². Careful observations uponinitiation of this method demonstrated that hindpaws were exposed to UVthroughout this period.

Automated Von Frey Hair Testing.

Hindpaw mechanical withdrawal thresholds were determined by theautomated von Frey hair method, using a 0.5 mm diameter stainless steelfilament, part of an automated plantar touch stimulator (Ugo Basile,Modena, Italy). Relevant detail included pre-test acclimatization in aquiet room for 30 min, conducting the test at the same time of day andblinded observers. The stimulus was delivered to the hindpaw,automatically discontinued upon withdrawal, and its intensity recordedautomatically. 6-8 trials per animal were conducted, with equal exposureof both hindpaws, leading to an average withdrawal threshold. Resultsare reported as A-threshold, which was calculated by subtractingpost-treatment from pre-treatment measurements, expressed as % orrelative to 1.0.

Hargreaves' Test.

Hindpaw thermal (hot) withdrawal thresholds were determined by thewell-established Hargreaves' method, using an infra-red thermalstimulation device that delivers the stimulus from underneath thehindpaw combined with automatic shut-off upon withdrawal (Ugo Basile).Stimulation and measurements were conducted as described for von Freyhair testing. A cutoff of 20 sec was set to prevent tissue damage.

Formalin-Induced Nocifensive Behavior.

Videos were read by blinded observers for the total amount of time eachmouse spent flinching or licking the injected hindpaw.

Mouse Tissue Processing for 1 μm Semithin Sections and ElectronMicroscopy.

Samples were fixed in 2% glutaraldehyde, 4% PFA, and 2 mM CaCl₂ in 0.05M sodium cacodylate buffer, pH 7.2, at room temperature for >1 h,dehydrated, postfixed in 1% osmium tetroxide, and processed for Eponembedding. Semi-thin sections (1 μm) were stained with toluidine blueand photographed with an Axioplan 2 microscope (Zeiss). For EM analysis,ultrathin sections (60-70 nm) were counterstained with uranyl acetateand lead citrate. EM images were taken with a transmission electronmicroscope (Tecnai G2-12; FEI) equipped with a digital camera (modelXR60; Advanced Microscopy Techniques, Corp.).

Mouse Tissue Processing for Immunohistochemistry.

Routine procedures were followed as described previously (Chen et al.,2009). Mice were perfused transcardially with 30 mL PBS, followed by 30mL 4% paraformaldehyde. Tissues, including the L5 DRGs (bilateral), andfootpad preparations, were dissected and post-fixed in 4%paraformaldehyde. Tissue blocks were further cryoprotected in 30%sucrose in PBS for 24-48 hours. For mouse TRPV4, keratin-specificantibodies, phospho-ERK, IL-6, IL-1β, CXCL5 and caspase-1, tissue wasprepared as frozen blocks and subsequently sectioned on a cryostat. ForCD68, CD15 (neutrophil elastase) and CD3, mouse skin was prepared by 2%PFA perfusion. Footpad and DRG sections (both at 6-10 μm) werethaw-mounted, blocked with 5% normal goat serum (NGS; Jackson), thenincubated overnight at 4° C. with the following primary antibodies:rabbit anti-TRPV4 (1:300; Abcam), mouse anti-keratin 14 (1:300; Abcamagainst C-terminal peptide beyond residue 850); rabbit anti-keratin 14(1:1000; Fuchs-Lab), rabbit anti-phosph-ERK1/2 (1:500; Cell signalingtechnologies), goat anti-IL-1β (1:800; Abcam), goat anti-IL-6 (1:200;Santa Cruz Biotechnology Inc); rabbit anti-caspase-1 (1:200; BiovisionResearch Products, CA); goat anti-mouse LIX/CXCL5 (1:200; R&D SystemsInc), anti-CD68, anti-CD25, and anti-CD3 (AbDSerotec). Immunodetectionwas accomplished with appropriate fluorescently-labeled secondaryantibodies (AlexaFluor595, AlexaFluor488-conjugated antibodies at 1:600;Invitrogen; for CD15 biotinylated secondary antibody from donkey, 1:400followed by rhodamine-streptavidine 1:250), or with peroxidase-linkeddetection reagents (for CD68) for 2 hours at room-temperature. Sectionswere rinsed, mounted, and cover-slipped with fluoromount (Sigma).Digital micrographs were obtained using a BX61 Olympus uprightmicroscope, also with a Zeiss LSM510 confocal, both equipped withhigh-res CCD camera, and acquired with constant exposure settings, usingISEE or Zeiss Zen software. Morphometric analysis was conducted usingImageJ freeware (v1.45) with tailored regions-of-interest that sparedthe nuclear compartment. ImageJ was also used for determination of DRGsurface area.

Human Tissue Specimens Immunolabeling.

Human tissue was deparaffinized with xylene and ethanol series, thenwashed in PBS, and incubated at 80° C. for 20 min in Antigen Retrievalbuffer (Biogenex). Subsequently, specimens were washed in PBS.Endogenous peroxidase was blocked with 0.3% H₂O₂+0.01% sodium azide inPBS for 10 min at room temperature, followed by washing steps in PBS.Blocking was performed in 5% normal horse serum+0.3% Triton-X-100 in PBSfor 1 hour at room temperature. Primary antibodies (anti-TRPV4, Abcam,same as for mouse, 1:8,000; anti-ET1; anti-IL-1b as for mouse tissue)were incubated overnight at 4° C. in Ventana Antibody dilution buffer(Fisher). After washing in PBS, specimens were incubated withbiotinylated donkey-anti-rabbit secondary (Vector, BA-1000), in dilutedblocking buffer for 30 min. After washing with PBS, Avidin Biotin blockwas applied (Vector, PK4000) for 30 min at room temperature, and thepositive immunoreactivity was visualized with DAB (Fisher, NC 9567138).After washing in water, hematoxylin was used to counterstain nuclei.Tissues were washed, dehydrated, and then mounted in Permount (Fisher).For morphometric quantificaton of TRPV4, IL-1β, and ET1, five sectionsfrom each patient and healthy volunteers (n=3/group) were examined at amagnification of ×20 and photographed. The entire section wasdigitalized using Leica software, and analyzed using ImageJ. Forquantification, DAB and HE staining in 3 randomly selected epidermalregions (3.5×1.25 inches) of each image were separated using the IsoDatathresholding method in the Color Threshold Plugin. Relative signalintensities were calculated from background-corrected measurements.Values are expressed as mean of averages determined from five sectionsper patient. Quantification of human skin tissue for TRPV4, ET1, andIL-1β was performed from acute photodermatitis as compared to healthyskin (n=3 per group). Quantification of various subforms of chronicphotodermatitis as compared to acute photodermatitis and healthy skin iscurrently under active study. More final results await availability andproper staining of at least 3 cases per subgroup of human chronicphotodermatitis.

Primary Mouse Keratinocyte Cell Culture.

The epidermis was separated from the dermis by a 1-hour dispase (BDBiosciences) treatment. Then the keratinocytes were dissociated from theepidermis using trypsin. Keratinocytes were plated on collagen coateddishes or glass or quartz coverslips and grown in keratinocyte serumfree media (Gibco) supplemented with bovine pituitary extract andepidermal growth factor (EGF) (R&D Systems), 100 pmol cholera toxin(Calbiochem, San Diego, Calif., USA) and 1× antibiotics/antimycotics(Gibco), in an incubator at 5% CO₂ and 37° C.

UVB-Stimulation of Cultured Keratinocytes; Calcium Imaging.

Ca++ imaging of mouse 1° MK in response to chemical activation of TRPV4was conducted after loading with 2 μM fura2-AM, following a ratiometricCa2+ imaging protocol with 340/380 nm blue light for dual excitation.Ratios of emissions were acquired at 0.5 Hz. ΔR/R0 was determined as thefraction of the increase of a given ratio over baseline ratio, dividedby baseline ratio. For stimulation of cells with UVB, where fura-2 wasnot suitable because of the proximity of stimulation with 340/380 nm and295 nm, 2 μM fluo4-AM was used instead. Ca++ imaging was carried out at488 nm excitation, acquisition of emissions at 0.5 Hz, expressed asΔF/F0. In the custom-built UV optical system, UV LEDs were capped with aball lens, a transparent optical window in the shape of a hemisphericallens (FIG. 2B). The LED output optical beam focused at 15-20 mm from thelens, with a spot diameter of approximately 1.5-2.0 mm (FIG. 2C). Theelectrical power supply for the UV LEDs was a surface mount component onthe printed circuit board, which had a steady state 20 mA current outputthat was controlled by an external switch. The thermal equilibrationstage was set for physiological temperature. We confirmed thenon-thermal nature of UVB stimulation using the customized 295 nm LEDdevice in a dedicated experiment (FIG. 2D), thus confirming the specificmodality of stimulation as UVB.

Keratinocyte UV Irradiation Using 295 nm LED and Immunocytochemistry.

Mouse keratinocytes were cultured on collagen coated quartz coverslipsand then stimulated from the bottom using the previously mentioned UVoptical system using the 295 nm LED. 24 hours later the cells were fixedin 4% formaldehyde in PBS for 20 minutes, permeabilized with 0.1% TritonX-100 in PBS for 10 minutes, washed, then blocked in 10% normal goatserum in PBS for 45 minutes. Coverslips were incubated overnight withprimary antibody mouse anti-ET1 (1:200; Abcam), washed three times inPBS and incubated with secondary antibody for 2 hours at 25° C.Coverslips were washed three times in PBS, once with double-distilledH₂O. Digital images were captured using a 40× immersion lens on the BX61Olympus upright microscope. Morphometric analysis was conducted usingImageJ freeware with tailored regions-of-interest.

Determining UVB Permeation of the Skin.

First, the spot size of the UV input optical beam from a LED (UVTOP-295UV) was estimated, as shown in FIG. 16A, using the razor-edge opticalspot occlusion method (results shown in FIG. 16B). The UV LED waspowered with 20 mA of current, resulting in 500 μW of optical power in acircular focal spot 1.5 mm in diameter, with 70% of the total flux in a0.5 mm beam radius. The UV optical power transmitted through the samplewas detected by a Hamamatsu S127-66BR UV detector, and the output of thephotodetector was measured using a Keithley 236 source measure unit.Next, the foot-pad epidermis of a mouse was measured for UV transmissionby placing it on a quartz coverslip and exposure to the UV beam. TheGSK205 was administered to the foot-pad skin in an alcohol and glycerolsolution as for the experiments shown in FIG. 12A. The vehicle-controlgroup was treated with the alcohol and glycerol solution only. Anothercontrol group consisted of a commercially-available SPF100 preparationsunscreen in form of a cream, which was applied similar to thevehicle-control. The data for the GSK205 and sunscreen was normalized tothis control data.

Western Blotting.

Samples were separated by SDS-PAGE, and transferred to PVDF membranes(Bio-Rad). Membranes were blocked with dry milk, then probed withprimary antibodies (rabbit anti-TRPV4 (immunogen=final C-terminalepitope of TRPV4 as for immunolabeling), Alomone; anti-caspase-1,Biovision; mouse anti-β-actin, (clone AC-5) Abcam; mouse anti-β-tubulin,Iowa Hybridoma bank), followed by horseradishperoxidase-conjugatedsecondary antibodies (Jackson Immunoresearch). Secondary antibodies weredetected using Supersignal West Dura Extended Duration substrate(Amersham).

Acute Pancreatitis Mouse Model.

C57BL/6J male mice 8-10 weeks of age were subjected to acutepan-creatitis by intraperitoneal injections of supramaximal doses ofcaerulein (50 μg/kg) every hour for a total of 6 h, as previouslydescribed in⁶¹. Control animals received 25% DMSO-saline solution byintraperitoneal injection every hour for 6 h. Compound 16-8 wasdissolved in this vehicle (10 mg/kg) and injected i.p. 30 min prior tothe first injection of caerulein. Animals were sacrificed 1 h after thelast injection. Blood was collected and pancreatic tissue was promptlyisolated, weighed for determination of pancreas wet weight/body weightratio. Samples of tis-sues were fixed overnight in 10% neutral-bufferedformalin, paraffin embedded and H&E-stained, or pancreatic tissue wasquickly frozen and assessed for myeloperoxidase (MPO) activity. Serumamylase, MPO and histologic evaluation were conducted as describedpreviously⁶¹.

Assessment of nocifensive behavior⁸: Mice were housed in individualcages and video-recorded during the entire experiment. Two mice at atime were observed. Linear movement was measured as one event when micepassed through the median plane of the cage. Analysis began immediatelyafter the first caerulein/vehicle injec-tion and continued until the endof the experiment. Results were expressed as the sum of the movementevents spanning the 6 h time-period following the first injection.

Cell Cultures.

N2a cells were used for directed expression of TRP ion channels asdescribed previously¹³. TRPV4-eGFP from rat was used, previously foundto respond to stimulation with GSK101 and hypotonicity in similar manneras native, non-fused TRPV4. All other channels were native channels frommouse, eGFP was co-transfected. Stimulation of over-expressed TRPV4 wasconducted with GSK101 (5 nM), TRPV1 with capsaicin (10 μM), TRPV2 withhypotonicity (270 mosmol/L), TRPV3 with camphor (100 μM) and TRPA1 withmustard oil (100 μM). eGFP control-transfected N2a cells did not respondto these stimuli. Ca++ imaging was performed as describedpreviously^(13,14,34).

To visualize dose-response relationships, Hill plots were conductedusing the Igor Pro software program, which derived the plots based onthe following equation:

$y = {{Base} + {\left( {{Max} = {Base}} \right)/\left\{ {1 + \left\lbrack \frac{x\mspace{14mu}{half}}{x} \right\rbrack^{rate}} \right\}}}$

Primary porcine chondrocytes derived from femoral condyles of skeletallymature pigs were cultured and subjected to Ca++ imaging as describedpreviously^(19,32,62,63).

Astrocyte cultures were conducted following established protocols⁶⁴⁻⁶⁶.Astrocytes were prepared from Sprague Dawley rat embryos (E18). Briefly,the isolated cortices were minced, and then incubated with trypsin andDNase. Dissociated cells were suspended in Dulbeccco's Modified Eagle'sMedium (DMEM) supplemented with 10% fetal calf serum andpenicillin/streptomycin (100 U/ml and 100 μg/ml, respectively).Thereafter, cell sus-pensions were plated in 75 cm² tissue cultureflasks (10×10⁶ cells/flask) which were pre-coated with poly-L-lysine (10μg/ml). The cells were maintained in a 10%002 incubator at 37° C. After10-12 days, the media was removed and adherent cells were trypsinized(0.25%) and plated out onto coverslips for subsequent Ca++imaging^(34,67). >95% of the cells were found to express astrocytemarker, glial fibrillary acidic protein (GFAP)⁶⁸.

Cell Viability in Culture.

N2a cells were cultured in 96 well plates for 24-48 h. Cell viabilitystudies relied on metabolic capability monitored with the indicator dyeresazurin. Its reduction to resorufin (indicated by color change darkblue to pink) was monitored over time. Changes in absorbance at λ=570 nmwere recorded using a microplate reader (Molecular Devices).Metabolically active and viable cells shared the ability to reduceresazurin to resorufin whereas dead cells did not. Eight replicatecultures per experimental point were studied.

Assessment of Hepatic, Renal and Cardiac Function in Mice Treated with16- . . . Compounds.

Mice were treated i.p. with compounds 16-8 and 16-19 (10 mg/kg). Hepaticand renal integrity were analyzed by alanine amino-transferase- andcreatinine assays (Sigma), both relying on measurement of absorbance atλ=570 nm in 96-well micro-titerplates. 8 technical replicates per animalwere performed.

For heart rate assessment in mice treated with 16-8 and 16-19, animalswere fitted with two electrodes, one to the ear, via clip, one to therib-cage, using firm adhesive. Heart rate was monitored and analyzedusing axoscope and clampfit 9.2 software (Molecular Devices)

Liquid Chromatography—Tandem Mass Spectrometry (LC-MS/MS).

Mice were treated with 10 mg/kg i.p. of the respective inhibitor.Post-euthanasia harvested tissue was frozen in liquid nitrogen andstored at −80° C. for further analysis.

Frozen tissue samples were partially thawed and cut into 1 mm slices,5-15 mg tissue, 2-fold excess water (mass/vol.), 6-fold excessacetonitrile (16- . . . compounds) or methanol (GSK205) containingappropriate amount of internal standard, and 2.5 mm zirconia/silica bead(Biospec Products Inc.) were added to 500-μL polypropyl-ene (PP) conicaltube, homogenized in a Fast-Prep apparatus (Thermo-Savant) at speed “4”for 20 sec at room temp, and centrifuged at 13,600 g for 5 min at roomtemp. Depending on the expected concentration range of the measuredcompound, the supernatant was diluted 1/4-1/20 (in Mobile phase A, seebelow) and placed in autosampler for LC-MS/MS analysis.

The LC-MS/MS assay for 16- . . . compounds and GSK205 was developed onan Agilent 1200 series LC system interfaced with Applied Biosystems API5500QTrap, a hybrid triple quadruple-linear trap MS/MS spectrometer.

Analyst (version 1.6.1) software was used for mass parameters tuning,data acquisition, and quantification. LC column: 3×4 mm RP C18(Phenomenex, AJ0-4287) was operated at 35° C. Mobile phase A: 0.1%formic acid, 2% acetonitrile, in LC/MS-grade water; mobile phase B:acetonitrile; flow rate: 1 mL/min, 1:1 MS/MS:waste split. Run time was 4min. Diverter valve was used to send flow to MS/MS only between 1.2 and2.5 min. The elution gradi-ent was: 0-0.5 min, 1% B; 0.5-1.2 min, 1-95%B; 1.2-1.5 min, 95% B; 1.5-1.6 min, 95-1% B. Autosampler was oper-atedat 4° C.; injection volume was kept at 10-50 μL. Electrospray ionization(ESI) source parameters were: positive ionization mode, curtain gasflow=30, ionization potential=5500 V, temperature=500° C., nebulizinggas 1 flow=30, nebulizing gas 2=30, declustering potential=20 V. 16- . .. compounds and GSK205 were individually infused as 100 nM solutions in50% A/50% B at 10 μL/min flow rate and parameters optimized to providemaximal ion count for “parent” and collision-produced (“daughter”) MS/MSions. Parent/daughter quantifier [qualifier] ions utilized: GSK205(401.1/280[370]), 16-8 (400.1/279.1[91.1], 16-16 (387.1/280[105]), 16-18(415.2/280[370], 16-19 (414.1/279.2[91.1]. Standard (analyte ofinterest)/internal standard pairs utilized: GSK205/16-16, 16-8/16-16,16-18/GSK205, 16-19/16-8.

Calibration samples (n=6) were prepared by adding pure standard of themeasured compound to tissue homogenate (tissue+2-fold excess water,mass/vol) in the appropriate range needed for the particular dosingregime. Organs studied were analyzed alongside the study samples. Thefollowing are typical ranges used (the lower value representing also theLLOQ at 80% accuracy limit, all other calibrator levels at 85% accuracylimit): 0.38-6 nM (plasma), 6-100 nM (skin), 6-48 nM (heart), 7.5-120 nM(brain), 19-300 or 1500-24000 nM (liver), 56-900 or 1500-12000 nM(kidney), 500-8000 nM (fat). Peak integration, calibration, andquantification was performed within Analyst software. The response ofthe peak area standard/int. std. to nominal concentration was linearwith r=0.999 or better.

Patch Clamp Recordings.

Heterologously transfected N2a cells were subjected to patch clampelectrophys-iological recordings. Briefly, 24 h after transfection cellswere prewashed with extracellular fluid (ECF) which contained (in mM) 1MgCl₂, 10 Glucose, 10 HEPES, 145 NaCl and 2 CaCl₂) (pH 7.4, 310 mOsM).Cells were then incubated with or without TRPV4 inhibitors in ECF for 5min before whole cell recording. Cover slips were transferred to arecording chamber mounted on the stage of a Leica inverted microscopethat was equipped with fluorescent filters. Transfected cells wereidentified before patching by their green florescent color. Cells werepatched with a 2.5-3.0 MΩ glass electrode pulled from borosilicate glasscapillaries using pipette puller (Sutter instruments). The intracellularsolution contained (mM) 140 CsCl, 10 HEPES, 1 EGTA, 0.3 Na-GTP, 2Na₂-ATP, and 2 MgCl₂ (pH 7.4, 295 mOsM). Whole cell currents wererecorded using pclamp 9.2 software and Axopatch 200B amplifier(Molecular Devices). The cells were first clamped at −65 mV beforeapplying a 1 s voltage ramp from −110 mV to +120 mV. The voltage rampwas applied every 2 seconds for 15 to 20 sweeps. Capacitance wasmonitored throughout the experimental recordings. Reported data waswithin ±3 pF.

Example 2: Generation of an Epidermal-Specific, Tamoxifen-InducibleTrpv4 Null Mouse

To circumvent developmental issues that can arise in gene-targeted micewith ubiquitous deletions, we developed an inducible conditional systemto assess the roles of TRPV4 in UVBmediated skin irritation,inflammation, and sensory sensitization. Using mouse ES cells, we firstbuilt Trpv4lox/lox mice so that the sizable exon coding fortransmembrane domains 5, 6, and the interjacent pore loop was flanked byloxP elements. After crossing to FLPe mice to remove the selectionmarker, flanked by frt elements, these animals were mated with tamoxifen(tam)-inducible, Keratin-14 (K14)-CRE^(EK) transgenic mice. Theconstructs and genotyping are summarized in FIG. 1A-B.

We focused on adult (2 month) glandular mouse paw-pad skin for ouranalyses, as it more closely resembles human skin. Tamoxifen-inductionresulted in efficient knockdown of Trpv4 expression in skin epidermis,as judged by anti-TRPV4 immunolabeling, qRT-PCR and Western blotting(FIG. 3A). However, the gross and microscopic appearance of theskin/epidermis in tam-treated inducible Trpv4 knockout (iKO) mice wasnormal. Given the established dependence of skin renewal on keratinocyteCa++ signaling, we noted that, interestingly, Trpv4-knockdown resultedin no gross alterations in the skin/epidermis nor in the induction ofthe terminal differentiation-specific marker keratin-1 (K1), which isknown to be governed by elevated Ca++ influx suprabasally (FIG. 3A andFIG. 1C). Closer analysis of Trpv4-deficient skin revealed thatexpression of K14 was sustained suprabasally. This keratin is normallydown-regulated at the basal-to-suprabasal transition, concomitant withthe rise in Ca++ signaling and induction of terminal differentiation.

Taken together, despite these more moderate abnormalities, inducingTrpv4 knockdown in keratinocytes at age 8 weeks does not lead to grossinterference with cyto- and layer architecture of the epidermis.

Example 3: Nocifensive Behavior Elicited by UVB Exposure is Dependent onEpidermally-Expressed TRPV4

Underscoring the specificity of Trpv4 gene targeting, peripheral sensoryneurons innervating the footpad still showed robust expression of TRPV4(FIG. 1D). This enabled us to evaluate whether epidermalTrpv4-deficiency critically affects UVB-mediated nocifensive behaviors.For this purpose, we assayed two relevant submodalities—thermal andmechanical stimulation—and compared our iKO mice to pan-null Trpv4−/−and their wild-type (WT) controls (FIG. 3B). 48 hours after UV-exposure,both tamtreated iKO and Trpv4−/− mice displayed much lower sensitivityto noxious radiant heat (Hargreaves' test), also towards noxiousmechanical stimulation (using automated von Frey hair testing), thantheir respective controls. We concluded that epidermal-specific TRPV4deficiency is equivalent to global Trpv4 ablation in reducingUVB-induced behavioral sensitization to radiant heat and mechanicalstimuli, that is, in attenuating thermal and mechanical allodynia.

Further underscoring the importance of epidermal TRPV4 in regulatingnocifensive behavior, a good correlation existed between UV-sensitivityto thermal stimuli and the level of Trpv4 gene knockdown, particularlyat <0.45 the WT levels of Trpv4 mRNA (FIG. 3C). This indicated presenceof a threshold for Trpv4 knockdown to influence nocifensive behavior.Although the dose-responsiveness was less obvious in our mechanicalassay (not shown), we attributed this to the involvement of forced hindlimb (foot) movement in the assay, which will confound the stimulus. Bycontrast, the Hargreaves' assay applies a purely thermal cue whichbecomes noxious without involving confounding stimuli.

In order to assess the specificity of the injurious stimulus, we inducedirritation with foot-pad injections of formalin, eliciting thewell-established bi-phasic response. In this assay, conditionalepidermal knockdown of TRPV4 had no effect on direct peripheral chemicalirritation (phase I) or the early maladaptive neural response (phase II)(FIG. 3D). Taken together, these results suggested that the level ofepidermal Trpv4 knockdown is the determining factor for the degree ofattenuation of nocifensive behavior caused by UVB-irradiation. This isspecific because chemical irritant-induced nocifensive behavior is notaffected by epidermal Trpv4 knockdown.

In additional control experiments, Trpv4lox/+ heterozygous mice hadvirtually identical behavioral sensitization (similar to WT) in responseto UVB, irrespective of CRE-induction with tamoxifen or vehicle (FIG.1E). These findings exclude a functional role for CRE^(ER) on its ownand reiterate the specificity of our approach in targeting Trpv4ablation to keratinocytes. Also, Trpv4−/− skin was equally permeable toUVB as its WT counterpart.

Example 4: Activation Markers of Skin-Innervating Peripheral NeuronsSupport Behavioral Findings

In WT mice, the footpad is innervated by sensory neurons of the L5 DRG,which we examined by immunolabeling. TRPV4 expression was unchanged withfoot-pad exposure to UVB, an irritant cue known to sensitize innervatingneurons (FIG. 1D). Interestingly, while sensitization could be verifiedin control mice, it appeared to be absent in tam-treated iKO mice, asdocumented by labeling for phosphorylated ERK (pERK), a known marker ofsensory neuron activation in response to inflammation and irritation(FIG. 3E). Furthermore, size-measurements of pERK-expressing L5 DRGneurons revealed them to be small-to-medium size, suggesting theirpossible involvement in relay of noxious stimuli (FIG. 1F). Theseresults are in good agreement with the nocifensive behavior defects seenin our mice, and further underscore a role for epithelial-expressedTRPV4 in governing UVB-induced activation in skin-innervating DRGsensory neurons.

Example 5: UVB-Induced Skin Inflammation Depends Upon EpidermalExpression of TRPV4

To understand how loss of TRPV4 affects UVB-induced skin, we performedlight microscopy and ultrastructural analyses (FIG. 4A and FIG. 5A-C).In response to UVB, robust signs of inflammation appeared within controlskin, as evidenced by intra-epidermal infiltrates of granulocytes. Withthe epidermis, focal blistering occurred, accompanied by extensivevacuolization. In skin of tam-treated iKO mice with incompletetargeting, inflammatory changes were still observed and were perhapsmoderately less severe. In striking contrast, however, in skin areaswhere conditional epidermal ablation of Trpv4 was complete, no signs ofinflammation or blistering were seen. These data demonstratedconvincingly that epidermal TRPV4 is necessary for skin to mount apro-inflammatory response to UVB exposure. Moreover, since theinflammation involved immune cells and the conditional knockout wasspecific to epidermis, the data further highlight the importance ofepidermal-inflammatory cell crosstalk in the response. Specifically,these data imply that in normal skin, the epidermal keratinocytetriggers inflammatory cell recruitment as part of a UVB response, andthat this circuitry is interrupted when epidermal TRPV4 is knocked down.

We next sought to identify the specific TRPV4-dependent epidermalsignals that occur in WT mice exposed to UVB, and the immune cellpopulations that respond. IL-6 was a suitable candidate for theepidermal signal since it is an established marker of skin epidermalactivation during UV dermatitis, and in addition, IL-6 is robustlyalgogenic. Indeed not only was IL-6 immunoreactivity observed in theUVB-exposed epidermis of control mice, but in addition, this robust IL-6upregulation was virtually eliminated in conditionally targeted as wellas pan-null Trpv4 knockout skin (FIG. 4B and FIG. 5D).

Both macrophages and neutrophils are known to contribute to thereduction of pain thresholds via their expression of a host ofproalgesic/algogenic mediators such as TNFα, IL-6, IL-8, proteases, andchemokines. As judged by immunostaining for CD68 (macrophages) and acell type-specific elastase (indicative of activated neutrophils, alsoknown to enhance nociception), UVB-induced infiltration of both of thesecell populations was markedly reduced in the skin of Trvp4-conditionalknockout mice (FIG. 4C-D). By contrast, the mast cell infiltrate wasunaffected (FIG. 5E), underscoring the specificity of macrophage andactivated neutrophil findings; T-cell count was not changed betweengenotypes either. Taken together, these findings showed that TRPV4expression by keratinocytes is critical for their ability to generateIL-6 and attract macrophages and activated neutrophils in response toUVB radiation.

Example 6: The UVB-Induced Ca++ Response in Primary Mouse Keratinocytesto UVB is Critically Dependent on Extracellular Ca++ Influx ThroughTRPV4

To further dissect the underlying mechanisms involved, we built acustomized device for specific and narrow-band UVB stimulation ofprimary mouse epidermal keratinocytes (1° MK) cultured in vitro (FIG.6A-B and FIG. 2A-D). This allowed use of the Ca++ sensitive dye, fluo-4,and assessment of 1° MK's Ca++ dynamics following UVB exposure (FIG.6B). The elicited Ca++ signal was obliterated by a UV-refracting glasscoverslip, underscoring the strict dependence of the calcium response onUVB (FIG. 6C and FIG. 2A).

Next, we asked whether the UVB-mediated Ca++ response is dependent onextracellular Ca++, and recorded affirmative findings by sequentialexposure to first UVB, then Ca++ (FIG. 6D). This finding prompted us todirectly query the role of TRPV4 in the UVB-mediated Ca++ response.Indeed, 1° MK from Trpv4−/− mice exhibited a greatly diminished responserelative to their WT counterparts (FIG. 6E). Moreover, when a selectivesmall molecule-compound, GSK205 (Vincent and Duncton. Current Topics inMedicinal Chemistry 2011, 11, 2216-2226), was used to block TRPV4channel function, WT 1° MK showed a very similar response to that ofTrpv4−/− 1° MK (FIG. 6F).

In view of the known robust expression of TRPV3 in keratinocytes(Moqrich et al., 2005; Peier et al., 2002), we also addressed TRPV3'srole in UVB-mediated Ca++ increase, but observed no effect with theTRPV3-selective inhibitor, IPP (FIG. 6G). The same dose of IPP waseffective in inhibiting camphor-evoked Ca++ transients (FIG. 2E),validating the negative result.

Together, our experiments indicated that UVB exposure to the epidermiselicits the influx of extracellular Ca++ through TRPV4 and not TRPV3channels. Since both channels were present, the data further suggestedthat TRPV4 channels are selectively activated by UVB light. We obtainedcorroborating findings by chemically activating TRPV4 with GSK101, whichcan directly stimulate TRPV4 in WT 1° MK. The GSK101-mediated responsewas dependent upon external Ca++ and was eliminated by the TRPV4inhibitor GSK205 (FIG. 2F). These findings showed that direct chemicalchannel activation of TRPV4 shares critical properties of UVB-evokedCa++ dynamics.

To assess whether TRPV4 is sufficient for the UVB-evoked Ca++ influx, weintroduced high levels of TRPV4 into HEK293 epithelial cells. TRPV4expression endowed these cells with the ability to generate robust Ca++signaling in response to UVB (FIG. 2G). Moreover, if they werepre-exposed to GSK205, the response was blocked. Thus, heterologousTRPV4 expression is sufficient for UVB radiation to cause a cellularCa++ transient.

Example 7: Elevated Endothelin-1 is a Critical Epidermal Effector of theUVB-TRPV4-Ca++ Response

The UVB-TRPV4-Ca++ response depended on upon phospholipase-C (PLC), asit was virtually eliminated by the specific PLC inhibitor, U73122 (FIG.6H). The reliance of TRPV4 activation upon PLC signaling suggested thatPLC's respective lipid products, such as IP3, might be involved. It alsohinted at possible involvement of G protein-coupled receptor signaling.

Using a candidate approach, we focused on endothelin receptors [ET(R)],which are known to be expressed in skin keratinocytes. ET(R)s wereparticularly good candidates since they functionpro-algesic-/algogenically, and their cognate peptide ligand,endothelin-1 (ET1), is elevated when keratinocytes are exposed to UVB.When our 1° MK were exposed to ET1, they exhibited a significantincrease in their UVB-induced Ca++ signaling (FIG. 7A(i)). This responsewas dependent upon TRPV4, as it was greatly diminished by GSK205.Consistent with this result, ET1 augmented GSK101-evoked Ca++ signaling(FIG. 8A).

We next blocked ET1 secretion by applying the proendothelinconvertase-inhibitor, CGS35066. This inhibitor significantly diminishedUVB-induced Ca++ signaling in 1° MK (FIG. 7A(i)). Since ET1's enhancingeffect on Ca++ signaling was dependent upon TRPV4, and the UVB-Ca++response was significantly sustained by ET1 secretion in vitro, weposited that autocrine/paracrine signaling involving ET1 may function toactivate its cognate receptors, ET-R(A) and ET-R(B). In good agreementwith this notion, ET-R inhibitors markedly attenuated the Ca++ signal inresponse to UVB and ET1 co-exposure. Interestingly, antagonism ofET-R(A) eliminated later phases of the Ca++ response, while leaving theinitial rise unaffected; by contrast, antagonism of ET-R(B) convertedthe UVB-Ca++ response into a more protracted one (FIG. 7A(ii)).Co-application of both inhibitors completely eliminated the UVB-inducedCa++ response by 1° MKs (FIG. 7A(iii)).

Taken together, these findings indicate that UVB-mediated ET1 secretionis a significant contributor to the UVB-TRPV4-Ca++ response. The datafurther suggest that independently from UVB's other effects, ET1-ET(R)co-signaling can amplify a TRPV4-dependent Ca++ response. In support ofthis notion, the UVB-Ca++ response could be recapitulated by omittingUVB-exposure and instead co-treating 1° MKs with ET1 and the selectiveTRPV4 activator 4α-PDD. Moreover, this response was significantlyattenuated by ET(R)-A inhibition, and greatly diminished by ET(R)-Binhibition (FIG. 7B). Selective antagonism of ET(R)s led to attenuationfor both compounds, yet showed a slight difference to the patternobserved with UVB, namely co-dependency for both ET(R)s for UVB, andrecruitment of ET(R)-B more than -A for 4α-PDD. Given the pleiotropiceffects of UVB, these minor differences were not surprising, andoverall, the results provided compelling support for the interdependenceof TRPV4 and ET(R) signaling in the response.

Interestingly, un-stimulated 1° MKs produced appreciable levels of ET1,secretory behavior which was dependent upon TRPV4 and PLC (FIG. 8B). Wetested whether UVB causes ET1 upregulation in a TRPV4-dependent manner.Given the exquisite wave-length dependence of ET1 expression in 1° MKs,we resorted to the UVB-LED device as for Ca++ imaging (FIG. 6A and FIG.2A-D). UVB-exposed 1° MKs showed increased ET1 immunolabeling, which wasdiminished when cells were preincubated with TRPV4-inhibitors, GSK205and RN1734 (FIG. 8C-D). Consistent with its effects on UVB-evoked Ca++transients, PLC-inhibitor U73122 also dampened ET1 expression. Together,these findings suggested a limited feed-forward mechanism that involvesTRPV4-dependent increase of ET1 expression in response to UVB, andautocrine/paracrine signaling via ET(R)s. This leads to TRPV4-dependentCa++ signaling, which in turn amplifies ET1 signaling in aparacrine/autocrine fashion.

To assess the physiological relevance of our findings in vivo, weexposed paw-pads to UVB. An interesting time course of Edn1 mRNAexpression was apparent in paw-pad skin of WT mice where it peaked after120 min and relented at 24 hours, but remained significantly elevated.In contrast, there was no regulated expression of Edn1 in paw-pad skinof Trpv4−/− mice. These findings suggest a more direct regulation ofEdn1 gene-expression by TRPV4 in response to UVB. ET1 was readilydetected in control epidermis but reduced in TRPV4-deficient epidermis(FIG. 7C). TRPV4 was also critical for the facilitatory effect of ET1 onnocifensive behavior, as judged by the finding that the withdrawalthresholds in response to von Frey hair stimulation were significantlylowered in control but not Trpv4−/− mice, both pan- and conditionalnull, in response to subepidermal ET1 injections (FIG. 7D). This resultsuggested that ET1's pro-algesic/algogenic effect is fully dependent onTRPV4 in keratinocytes. Although previous studies had shown that ET1 issufficient to elicit nocifensive behavior, the elimination of ET1'spro-algesic/algogenic effect in our Trpv4-conditional null mice wasunexpected given that both ET(R)s and TRPV4 are expressed by sensoryafferents, which were unaffected in our tam-treated iKO mice.

Example 8: UVB-Induced Activation of Inflammasomes by KeratinocytesDepends Upon TRPV4

Another signaling mechanism linking UVB exposure to inflammation andnociception in keratinocytes is the inflammasome, a large multiproteincomplex that assembles in response to infection and other cellularinjury, and triggers an inflammatory cascade culminating incaspase-activation and production of cytokines IL-1 and IL-18.Previously, its formation was shown to depend upon Ca++ signaling,prompting us to query the dependence of inflammasome activation onTRPV4. Indeed, although caspase-1 was upregulated in UVB-treated controlskin, this was largely eliminated in Trpv4−/− and greatly attenuated intam-treated iKO skin (FIG. 9A-B). Moreover, even though caspase-1cleavage (activation) was readily detected by Western blotting oflysates from WT 1° MK exposed to UVB, caspase-1 expression was lackingin Trpv4-null counterparts, even without UVB (FIG. 9C).

Similarly, upregulation of the pro-algesic inflammasome product IL-1βwas readily detected in response to UVB treatment of control skinepidermis but not TRPV4-deficient epidermis. This was demonstrated notonly by immunolabeling but also by measuring IL-1β levels in paw-padedema interstitial fluid (FIG. 9D-F). Together, these results establisha fundamental importance of TRPV4 in keratinocyte-mediated activation ofinflammasomes, which enhance caspase-1-mediated proteolytic cleavage ofpro-IL-1β to form active IL-1β.

Related to IL-1β secretion by skin in response to UVB, we querieddependence of CXCL5 on Trpv4. CXCL5, whose expression is dependent uponIL1β/IL1R1 signaling, has recently been reported to function in aproalgesic/algogenic manner in keratinocytes in response to UVB inrodents and humans. Consistent with the reliance of inflammasomefunction and IL-1β expression on TRPV4, we found that similarly,UVB-induced proalgesic/algogenic CXCL5 upregulation is also dependentupon keratinocyte-derived TRPV4 (FIG. 9G-H).

Example 9: Clinical Relevance of Epidermally-Derived TRPV4 inTransmitting Nociceptive Responses to UVB Exposure

Interestingly, TRPV4 was significantly increased in the epidermis ofhuman patients with UV photodermatitis (FIG. 10A and FIG. 11 ). Ascompared to healthy skin controls, a robust increase was also seen forET1 and IL-1β immunostaining in acute photodermatitis (FIG. 10A-B).These findings suggest that TRPV4 is also likely to be involved inUVB-induced photodermatitis, one of the various responses of human skinto damaging UV radiation.

In view of the observed impact of epidermal-specific TRPV4-deficiency onmouse nociception in response to UVB, and because of the unambiguouseffects of selective TRPV4 blockers on 1° MK in vitro, we tested thepossible clinical relevance of our findings. For this purpose, wetopically applied TRPV4 inhibitor GSK205 to WT mouse skin andsubsequently exposed animals to UVB (FIG. 12A). While solvent-treatedcontrol mice displayed a normal thermal hypersensitivity response, micetreated with 1 mM GSK205 showed a ˜24 hour delay in sensitivity, andincreasing the dose to 5 mM GSK205 resulted in a sustained attenuationof thermally-evoked nocifensive behavior. Of note, this treatment alsoresulted in a significantly reduced sensitivity of mice to von Frey hairmechanical stimulation.

To assess specificity of the external-topical treatment, we applied 5 mMGSK205 to Trpv4−/− mice vs. vehicle control (FIG. 12A). Nocifensivebehavior did not show any inter-group differences, yet was significantlydifferent from pre-stimulation thresholds. Based upon these results,topical treatment with 5 mM GSK205 in vivo did not elicit off-targeteffects on other channels or signaling pathways that might measurablyinfluence withdrawal behavior. Although GSK205-mediated antagonism ofmacrophage and neural TRPV4 cannot be excluded, the epidermis is theinitial target of topically applied drugs, and the effects we measuredwith 5 mM GSK205 were consistent with those we observed in Trpv4tam-induced iKO skin.

Histopathology of GSK205-topically-treated skin showed hallmarks ofUVB-photodermatitis in vehicle-treated paws, strikingly contrasting toGSK205-treated animals (5 mM), whose paws showed virtual elimination ofinflammation (FIG. 14A). In view of TRPV4-dependence of ET/Edn1expression in skin, we measured Edn1 mRNA abundance in GSK205 vs.vehicle treated paw-pad skin. We detected an early upregulation invehicle-treated mice at the 2 hour time-point which was stillsignificantly elevated over preexposure animals at 24 hours, resemblingEdn1 regulation and time-course in untreated WT mice (FIG. 14B, compareto FIG. 15 ). In striking contrast, and in keeping with histopathology,Edn1 mRNA-expression in GSK205 topically-treated skin was foundunchanged.

To validate these finding, we tested UVB absorption in GSK205-treatedpaw-pad skin, and whether GSK205 thus functions as sunscreen. Resultswere negative, as demonstrated by equal UVB permeation of GSK205-vs.vehicle-treated paw-pad skin, yet valid, as demonstrated by significantdecrease of UVB permeation with SPF100 sunscreen (FIG. 14CD and FIG.16AB). These results corroborate that effects of topically appliedGSK205 are caused by TRPV4 antagonism, likely by affecting TRPV4 inepidermal keratinocytes.

We also tested the response of down-stream UVB effector mechanisms toGSK205 treatment in vivo. In mouse skin, IL-1β was upregulated inresponse to UVB with vehicle treatment, yet failed to upregulate with 5mM GSK205 (FIG. 12B-C). This in vivo finding was recapitulated in 1° MKwhose UVB-induced increase in IL-1β secretion was completely eliminatedin the presence of 5 μM GSK205 (FIG. 12D). Comparably significant blockswere observed on CXCL5 and IL-6 upregulation (FIG. 13A-B).

Thus, the UVB-evoked signaling in the epidermis was reduced both whenTRPV4 was antagonized by topical application of specific small moleculeinhibitors, and when Trpv4 was targeted genetically in ourkeratinocyte-specific and inducible Trpv4 conditional null mice. Thissuggests ion channel function of TRPV4 to be the critical factor commonto both experimental approaches. Taken together, these findings renderselective TRPV4 blockers, such as GSK205, excellent candidates fortherapeutic approaches to reduce damaging inflammatory responses causedby UVB exposure in humans.

Example 10: Importance of TRPV4 for the Itch-Response

Behavioral studies show reduced scratching behavior in Trpv4 null micecompared to WT mice. Histamine (10%) was injected intracutaneously intothe cheek of C57b/6 control (WT) or Trpv4 null mice (n=6 per group).Over 30 min, Trpv4 null mice showed a significant reduction ((p<0.01)t-test) as compared to WT mice (FIG. 17 ). The results demonstrated thatsimilar to TRPV1 and TRPA1, TRPV4 is involved in itch, and that blockersof TRPV4 activation may be beneficial for the treatment of itch.

Example 11: Evidence for the Role of TRPV4 in Itch

It was further examined whether TRPV4 has a role in itch using mice witha selective TRPV4 deletion and Compound 48/80. Compound 48/80 (availablefrom Sigma-Aldrich, St. Louis, Mo.) is a well-established pruritogen andelicits histamine-dependent itch by degranulating mast-cells. Mice wereused in which TRPV4 channels had been selectively deleted in skinkeratinoctyes by gene targeting, and the targeted allele was induced byfeeding of tamoxifen, as detailed in Example 2. Wild-type mice were usedas a control.

Compound 48/80 (100 micrograms in 50 μL) was injected retro-auricularlyinto the mice, and mouse scratch behavior in response thereto wasmonitored. Results are shown in FIG. 18 . Compared to control mice,scratch behavior over 30 min was significantly reduced for mice in whichTRPV4 channels had been selectively deleted in skin keratinoctyes.

The results provided additional evidence for the role of TRPV4 in itch,and in particular, dependence of itch on the TRPV4 ion channel expressedin keratinocytes of the skin. The results clearly indicated that TRPV4in skin epithelial cells (keratinocytes), and not sensory neurons orimmune-related or allergy-related cells, is the critical site of TRPV4expression and function in histamine-dependent itch. These findings alsosuggested that topical targeting of TRPV4 channels may be successful incombating itch.

Example 12: Overall Preparation of Compounds

The general scheme for preparation of compounds 16-8, 16-12c, 16-13,16-14, 16-16, 16-18, and 16-19 is below, with the following reagents andconditions for each step: (i) K₂CO₃, CH₃CN; (ii) Zn, MeOH, 12 M HCl;(iii) 1,1′-Thiocarbonyldiimidazole (iv) 7 M NH₃ in MeOH; (v) EtOH,reflux:

Step (i) General Procedure for the SN2 Displacement of 4-NitrophenethylBromide.

Powdered, oven-dried K₂CO₃ (1.5 eq.) and the amine (1.5 eq.) were addedsequentially to a room temperature solution of the bromide (0.33 M) inanhydrous CH₃CN. The reaction mixture was heated to 80° C. (oil bathtemp) until analysis of the reaction mixture by LCMS indicated completeconsumption of the bromide (˜6-18 hours). The mixture was cooled to roomtemperature and diluted with brine (two volume equivalents). Theresulting emulsion was extracted with EtOAc (2× one volume equivalent).The combined extracts were added to silica gel (mass of silica gel=2×mass of starting bromide) and the mixture was concentrated to drynessunder reduced pressure. Flash column chromatography (RediSepRf SiO₂,100% CH₂Cl₂→5% MeOH in CH₂Cl₂) gave the product as a brown to amber oil.The yield of the intermediates 13a-d (the tertiary amines formed in step(i)) are presented in Table 1.

TABLE 1 Yield of tertiary amines 13a-d formed in step (i) IntermediateNo. R n yield 13a Me 0 17% 13b Me 1 49% 13c Me 2 42% 13d Et 1 15%

Step (ii) General Procedure for the Nitro to Aniline Reduction.

A solution of the nitro compound (0.5 M in MeOH) was cooled in anice-NaCl bath. Zinc dust (4.5 eq.) was added in one portion followed bydrop wise addition of 12 M HCl (4.5 eq.) over 2-3 minutes. After 1 hour,the cooling bath was removed, and the reaction mixture was allowed tostir over night at room temperature. The following morning, the mixturewas cooled in an ice-NaCl bath once again and 30% aqueous NaOH was addeddrop wise until pH 14 (universal indicating pH paper) was reached. Themixture was diluted with CH₂Cl₂ (five volume equivalents) and stirredfor 5 minutes. After this time, insolubles were removed at the vacuum,and the filter cake was washed with CH₂Cl₂ (2×25 mL). The organic phaseof the filtrate was separated, washed with brine (100 mL), and dried(MgSO₄). The drying agent was removed by filtration. Silica gel (˜5 g)was added, and the filtrate was concentrated to dryness under reducedpressure. Flash column chromatography (RediSepRf SiO₂, 100% CH₂Cl₂→5%MeOH in CH₂Cl₂) gave the product as a clear, amber oil. The yield of theintermediates 14a-d (the anilines formed in step (ii)) are presented inTable 2.

TABLE 2 Yield of anilines 14a-d formed in step (ii) Intermediate No. R nyield 14a Me 0 75% 14b Me 1 84% 14c Me 2 97% 14d Et 1 85%

Steps (iii) and (iv) General procedure for thiourea formation. Asolution of the aniline (0.22 M) in anhydrous CH₂Cl₂ was added drop wiseover 2-5 minutes to an ice-NaCl bath cooled solution of1,1′-thiocarbonyldiimidazole (2 eq., 0.15 M) in anhydrous CH₂Cl₂. After15 minutes, the cooling bath was removed and the reaction mixture wasstirred at room temperature until analysis by TLC (5% MeOH in CH₂Cl₂)indicated complete consumption of the starting aniline. The mixture wascooled once again in an ice bath and 7 M NH₃ in MeOH (10.5 eq.) wasadded drop wise over 2-5 minutes. The bath was removed and the mixturewas stirred over night at room temperature. Silica gel (mass of silicagel=2× mass of starting aniline) was added and the mixture wasconcentrated to dryness under reduce pressure. Flash columnchromatography (RediSepRf SiO₂, 100% CH₂Cl₂→10% MeOH in CH₂Cl₂) gave thepure thiourea. The yield of the intermediates 15a-d (the thioureasformed in steps (iii)-(iv)) are presented in Table 3.

TABLE 3 Yield of thioureas 15a-d formed in steps (iii)-(iv) IntermediateNo. R n yield 15a Me 0 99% 15b Me 1 96% 15c Me 2 88% 15d Et 1 67%

Step (v) General Procedure for Thiazole Formation.

A mixture of the thiourea (0.1 M) in EtOH and the α-bromoacetophenonederivative (1.1 eq.) was heated to 75° C. (oil bath temperature) untilanalysis by TLC (5% MeOH in CH₂Cl₂) indicated complete consumption ofthe thiourea. Silica gel (mass of silica gel=2× mass of startingthiourea) was added, and the mixture was concentrated to dryness underreduced pressure. Flash column chromatography (RediSepRf SiO₂, 100%CH₂Cl₂→10% MeOH in CH₂Cl₂) gave the pure thiazole hydrobromide. Theyield of the final products 16-8 to 16-19 (the thiazole hydrobromidesformed in step (v)) are presented in Table 4.

TABLE 4 Yield of thiazole hydrobromides 16-8 to 16-19 formed in step (v)Compound No. R n aryl yield 16-8  Me 1 phenyl 56%   16-12c Me 23-pyridyl 82% 16-13 Me 1 4-pyridyl 83% 16-14 Me 1 2-pyridyl 94% 16-16 Me0 3-pyridyl 98% 16-18 Et 1 3-pyridyl 31% 16-19 Et 1 phenyl 93%   16-43CMe 1 3-pyridyl 52%

Example 13: Preparation of Compounds 16-8 and 16-19

Compound 16-8. Compound 16-8 was prepared as detailed in Example 11.Briefly, a suspension of the bromide (5.01 g, 21.8 mmol), N-benzylmethylamine (4.2 mL, 33 mmol, 1.5 eq.) and K₂CO₃ (4.6 g, 33 mmol, 1.5eq.) in anhydrous CH₃CN (65 mL) was heated to 80° C. (oil bath temp) for18 hours, after which time the starting material was nearly complete.The mixture was cooled to room temperature and diluted with brine (120mL). The resulting emulsion was extracted with EtOAc (2×60 mL). Thecombined extracts were added to silica gel (˜10 g) and the mixture wasconcentrated to dryness under reduced pressure. Flash columnchromatography (RediSepRf SiO₂ (120 g), 100% CH₂Cl₂→5% MeOH in CH₂Cl₂)gave the product as a clear, dark orange oil (2.91 g, 49%). ¹H NMR(CDCl₃, 400 MHz): 8.13 (d, J=8.4 Hz, 2H), 7.32 (d, J=8.4 Hz, 2H),7.30-7.22 (m, 5H), 3.55 (s, 2H), 2.91 (t, J=6.8 Hz, 2H), 2.68 (t, J=6.8Hz, 2H), 2.29 (s, 3H). ESIMS: m/z 271 [(M+H)+].

A solution of the nitro compound (2.8 g, 10.4 mmol) in MeOH (20 mL) wascooled in an ice-NaCl bath. Zinc dust (325 mesh, 3 g, 4.5 eq.) was addedfollowed by drop wise addition of 12 M HCl (3.8 mL, 4.5 eq.) over 2-3minutes. After 1 hour, the cooling bath was removed and the reactionmixture was allowed to stir over night at room temperature. Thefollowing morning, the mixture was cooled in an ice-NaCl bath once againand 30% aqueous NaOH was added drop wise until pH 14 (universalindicating pH paper) was reached. The mixture was diluted with CH₂Cl₂(100 mL) and stirred for 5 minutes. After this time, insolubles wereremoved at the vacuum and the filter cake was washed with CH₂Cl₂ (2×25mL). The organic phase of the filtrate was separated, washed with brine(100 mL) and dried (MgSO₄). The drying agent was removed by filtration.Silica gel (˜5 g) was added and the filtrate was concentrated to drynessunder reduced pressure. Flash column chromatography (RediSepRf SiO₂ (120g), 100% CH₂Cl₂→5% MeOH in CH₂Cl₂) gave the product as a clear, amberoil (2.1 g, 84%). ESIMS: m/z 241 [(M+H)+]. This material was used in thenext step without further analysis or purification.

A solution of the amine (2.1 g, 8.7 mmol) in anhydrous CH₂Cl₂ (40 mL)was added dropwise over 2-5 minutes to an ice-NaCl bath cooled solutionof 1,1′-thiocarbonyldiimidazole (95%, 3.1 g, 17.4 mmol, 2 eq.) inanhydrous CH₂Cl₂ (120 mL). After 15 minutes, the cooling bath wasremoved and the reaction mixture was stirred at room temperature for 1.5hours after which time analysis by TLC (5% MeOH in CH₂Cl₂) indicatedcomplete consumption of the starting aniline. The mixture was cooledonce again in an ice bath and 7 M NH₃ in MeOH (13 mL, 91 mmol, 10.5 eq.)was added dropwise over 2-5 minutes. The bath was removed and themixture was stirred over night at room temperature. Silica gel (˜5 g)was added and the mixture was concentrated to dryness under reducepressure. Flash column chromatography (RediSepRf SiO₂ (120 g), 100%CH₂Cl₂→10% MeOH in CH₂Cl₂) gave the thiourea as an amber oil thatsolidified to a tacky residue upon standing (2.5 g, 96%).

A mixture of the thiourea (2.5 g, 8.3 mmol) and 2-bromoacetophenone (1.8g, 9.1 mmol, 1.1 eq.) in EtOH (80 mL) was heated to 75° C. (oil bathtemperature) for 20 minutes after which time analysis by TLC (5% MeOH inCH₂Cl₂) indicated complete consumption of the thiourea. Silica gel (˜5g) was added and the mixture was concentrated to dryness under reducepressure. Flash column chromatography (RediSepRf SiO₂ (120 g), 100%CH₂Cl₂→10% MeOH in CH₂Cl₂) gave the thiazole hydrobromide as a strawcolored glass (3.73 g, 93%).

Compound 16-19. Upon examination of the activity of GSK205 relative tocompounds 16-8 and 16-18, it seemed that removal of a nitrogen from thepyridyl group increased the potency of the TRPV4 antagonist, andaddition of an extra carbon to the nitrogen carbon side chain increasedthe potency of the TRPV4 antagonist. Compound 16-19 was formed and basedon the structures of 16-8 and 16-18. See FIG. 19 .

Compound 16-19 was prepared as detailed in Example 11. Briefly, asuspension of the bromide (5.01 g, 21.8 mmol), N-benzyl ethylamine (4.9mL, 33 mmol, 1.5 eq.) and K₂CO₃ (4.6 g, 33 mmol, 1.5 eq.) in anhydrousCH₃CN (65 mL) was heated to 80° C. (oil bath temp) for 18 hours, afterwhich time the starting material was nearly complete. The mixture wascooled to room temperature and diluted with brine (120 mL). Theresulting emulsion was extracted with EtOAc (2×60 mL). The combinedextracts were added to silica gel (˜10 g) and the mixture wasconcentrated to dryness under reduced pressure. Flash columnchromatography (RediSepRf SiO₂ (120 g), 100% CH₂Cl₂→5% MeOH in CH₂Cl₂)gave the product as an orange oil that solidified upon standing at roomtemperature (3.3 g, 53%). ¹H NMR (CDCl₃, 400 MHz): 8.13 (d, J=8.4 Hz,2H), 7.32 (d, J=8.4 Hz, 2H), 7.30-7.22 (m, 5H), 3.55 (s, 2H), 2.91 (t,J=6.8 Hz, 2H), 2.68 (t, J=6.8 Hz, 2H), 2.87 (q, J=6.8 Hz, 2H), 1.20 (t,J=6.8 Hz, 3H). ESIMS: m/z 285 [(M+H)+].

A solution of the nitro compound (3.0 g, 10.4 mmol) in MeOH (20 mL) wascooled in an ice-NaCl bath. Zinc dust (325 mesh, 3 g, 4.5 eq.) was addedfollowed by drop wise addition of 12 M HCl (3.8 mL, 4.5 eq.) over 2-3minutes. After 1 hour, the cooling bath was removed and the reactionmixture was allowed to stir over night at room temperature. Thefollowing morning, the mixture was cooled in an ice-NaCl bath once againand 30% aqueous NaOH was added drop wise until pH 14 (universalindicating pH paper) was reached. The mixture was diluted with CH₂Cl₂(100 mL) and stirred for 5 minutes. After this time, insolubles wereremoved at the vacuum and the filter cake was washed with CH₂Cl₂ (2×25mL). The organic phase of the filtrate was separated, washed with brine(100 mL) and dried (MgSO₄). The drying agent was removed by filtration.Silica gel (˜5 g) was added and the filtrate was concentrated to drynessunder reduced pressure. Flash column chromatography (RediSepRf SiO₂ (120g), 100% CH₂Cl₂→5% MeOH in CH₂Cl₂) gave the product as a clear, amberoil (2.3 g, 87%). ESIMS: m/z 255 [(M+H)+]. This material was used in thenext step without further analysis or purification.

A solution of the amine (0.110 g, 0.43 mmol) in anhydrous CH₂Cl₂ (2 mL)was added dropwise over 2-5 minutes to an ice-salt bath cooled solutionof 1,1′-thiocarbonyldiimidazole (95%, 0.162 g, 0.87 mmol, 2 eq.) inanhydrous CH₂Cl₂ (6 mL). After 15 minutes, the cooling bath was removedand the reaction mixture was stirred at room temperature for 1.5 hoursafter which time analysis by TLC (10% MeOH in CH₂Cl₂) indicated completeconsumption of the starting aniline. The mixture was cooled once againin an ice bath and 7 M NH₃ in MeOH (620 μL, 4.3 mmol, 10 eq.) was addeddropwise over 2-5 minutes. The bath was removed and the mixture wasstirred over night at room temperature. Silica gel (˜1 g) was added andthe mixture was concentrated to dryness under reduce pressure. Flashcolumn chromatography (RediSepRf SiO₂ (40 g), 100% CH₂Cl₂→10% MeOH inCH₂Cl₂) gave the thiourea as an amber oil that solidified upon standing(0.130 g, 97%).

A mixture of the thiourea (159 mg, 0.51 mmol) and 2-bromoacetophenone(0.113 g, 0.56 mmol, 1.1 eq.) in EtOH (5 mL) was heated to 75° C. (oilbath temperature) for 1 hour, after which time analysis by TLC (10% MeOHin CH₂Cl₂) indicated complete consumption of the thiourea. Silica gel(˜1 g) was added and the mixture was concentrated to dryness underreduce pressure. Flash column chromatography (RediSepRf SiO₂ (40 g),100% CH₂Cl₂→10% MeOH in CH₂Cl₂) gave the thiazole hydrobromide as astraw colored glass (0.165 g, 78%).

Example 14: Effect on TRPV4-Mediated Calcium Transport

Compounds (GSK205, 16-12, 16-13, 16-14, 16-18, 16-8, and 16-19) weretested for their effect on TRPV4-mediated calcium influx in N2a culturedcells with targeted expression of human TRPV4. Ca2+ imaging wasperformed according to Li et al. (Environ. Health Perspect. 2011, 119,784-93) and Moore et al. (Proc. Natl. Acad. Sci. U.S.A. 2013, 110,E3225-E3234). Briefly, Ca2+ imaging of primary mouse epidermalkeratinocytes (1° MK) in response to chemical activation of TRPV4 wasconducted after loading with 2 μM fura2-AM, following a ratiometricCa2+-imaging protocol with 340/380 nm blue light for dual excitation.Ratios of emissions were acquired at 0.5 Hz. ΔR/R0 was determined as thefraction of the increase of a given ratio over baseline ratio, dividedby baseline ratio. For stimulation of cells with UVB, where fura-2 wasnot suitable because of the proximity of stimulation with 340/380 nm vs.295 nm, 2 μM fluo4-AM was used instead. Ca2+ imaging was carried out at488 nm excitation, acquisition of emissions at 0.5 Hz, expressed asΔF/F0. TRPV4 was activated with 10 nM GSK101, a specific activator,which had no effect on RFP-transfected cells. Each of the six compoundswere added to a concentration of 2.5 μM, and its effect was observed.

Results are shown in FIG. 20 . The bar diagram to the left shows theeffects of 2.5 μM of the respective inhibitor (pre-incubation for 10min). The ordinate is the peak calcium concentration in the cells(average n>75 cells) in nM. All six compounds were effective ininhibiting TRPV4-mediated calcium influx, with 16-18 and 16-8 being themost potent. 16-19 did not show enhanced inhibition over 16-8 (data notshown). It was noted that GSK205 had a very low potency at thisconcentration in cultured cells with targeted over-expression.

Example 15: In Vivo Pain Model in Mice

Compounds were tested for their effect in reducing pain using an in vivopain model in mice. For mouse formalin-evoked irritant behaviormeasurements, mice were well-fed, well-rested, and tested at the sametime of day, at the same time-point of their circadian rhythm. They wereallowed to acclimate to a plexiglas chamber for at least 30 min beforetesting, and received 10 μL subcutaneous injection of 4% of formalin(diluted from an aqueous solution of commercial 37% formaldehyde withnormal saline (NS)) through a 30-gauge needle into the right whiskerpad,as further detailed in Luccarini et al. (J. Pain, 2006, 7, 908-914).Normal saline was used as control injection. After injection, mice wereimmediately placed back into the chamber and the rubbing behavior wasrecorded by a private consumer-type video-camera for a 45 minobservation period. The recording time was divided into 9 blocks of 5min, and a nociceptive score was determined per block by measuring thetime that the animals spent rubbing the injected area predominantly withthe ipsilateral fore-paw and rarely with hind-paw. This rubbing behaviorwith fore-paw is evoked by pain, which is distinct from itch behavior.Behavioral analysis was conducted by observers blinded to treatment.

To investigate the effects of the specific compounds GSK205, 16-8, and16-19 on formalin-induced nociceptive behavior, mice received a singlesubcutaneous injection of the compounds into the whiskerpad (10 μL,dissolved in 4% DMSO) 15 min before formalin injection. Control animalsreceived the same volume of NS, 4% DMSO.

Results are shown in FIG. 21 . FIG. 21A shows the time-course ofnocifensive behavior in response to whisker-pad injection of formalin toBL6 mice. Note the biphasic response and “clean” controls. FIG. 21Bshows quantitation of FIG. 21A, with n=10 animals per group. Note thesignificant increase in response to formalin injection. FIG. 21C showssimilar quantitation when compounds where pre-injected topically at 0.5mM/10 μL. This concentration had no effect on residual nocifensivebehavior in Trpv4−/− mice. Note the lack of effect of GSK205 at thisconcentration, yet a significant attenuation of nocifensive behavior, inparticular of the centrally caused second phase, in response to 16-8 and16-19. FIG. 21D shows the time course for the three compounds. GSK205,at this concentration, was not different from control, whereas 16-19 and16-8 significantly attenuated nocifensive behavior. Note the reductionto control levels with the less lipophilic 16-8 at the 45-mintime-point, and the reversal to control levels with the more lipophilic16-19 at this time-point. Also note that none of the compoundsinfluenced the first phase, which was caused by the direct irritationthat formalin causes on peripheral whisker-pad nerve endings.

Example 16: UVB-Exposure Evoked Nocifensive Behavior

Compounds were tested for their effect on nocifensive behavior (responseto pain) following UVB overexposure. Behavioral tests were performed toevaluate the decrease in withdrawal thresholds in response to mechanicalvon Frey hair or thermal stimuli applied to hind paws. The Von-Freyapparatus (Ugo Basile) applied a mechanical stimulus with a flexiblesteel wire from underneath the hind paw. The force leading to withdrawalwas determined. For the thermal stimuli test, paws were stimulated withheat from underneath applied by an infrared beam (Hargreave's testapparatus; Ugo Basile), and withdrawal latencies were recorded. Thewithdrawal thresholds were ascertained before and after UV exposure.Mice were exposed to UVB 3-5 days after the last application of tam/oil,using a Bio-Rad Gel Doc 2000 UV transilluminator (302 nm) for 5 min withan exposure of 600 mJ/cm². This represents 5-10 times the minimalerythema-inducing dose, in keeping with the rationale of inducingsunburn and studying sunburn-evoked pain.

Results are shown in FIG. 22 , demonstrating the effective topicaltreatment of UVB-overexposure evoked nocifensive behavior by compound16-8. Topically applied 16-8 was especially effective in reducing theresponse to pain following UVB exposure, whereas GSK205 at thisconcentration (0.5 mM in 40 μL), was as effective as vehicle(n=4/group).

Example 17: Effect on TRPA1

Compounds were tested for their effect on TRPA1. N2a permanent cellswere transfected with human TRPA1 cDNA, using a pcDNA3.1 expressionplasmid. They were co-transfected with eGFP-expressing plasmid, or, forcontrol, with eGFP plasmid only.

Vehicle-treated TRPA1-expressing cells showed a robust Ca2+ transiencein response to 60 μM AITC and also to 1 mM mustard oil, which are bothknown electrophilic TRPA1-activators. However, eGFP-expressingcontrol-transfected cells (no TRPA1) did not respond to theTRPA1-activator AITC. Cells were then pre-exposed to 5 μM of compounds16-8 and 16-19 for 10 min.

Results are shown in FIG. 23 . Averaged Ca2+ signal is shown from >75cells. The resulting Ca2+ transient evoked by 60 μM AITC was reducedby >80% upon administration of compound 16-8 or 16-19 at 5 μM,indicating appreciable TRPA1-inhibitory effects of both compounds.Therefore, compounds 16-8 and 16/8-18hy showed inhibitory activityagainst human TRPA1. For doses below or equaling 25 μM, GSK205 did notinhibit TRPA1 activity.

Example 18: Effect on TRPV1, TRPV2, and TRPV3

Compounds were tested for their effect on TRPV1, TRPV2, and TRPV3.Methods were similar to those described in Example 15. Briefly, N2acells were transfected with human TRPV1, TRPV2, or TRPV3.eGFP-transfection was used as control. For specific stimulation ofTRPV1, 5 μM capsaicin was used. For stimulation of TRPV2, hypotonicity(260 mosmol/L) was used, based on previous reports of TRPV2 beingosmotically responsive. N2a cells were not responsive to hypotonicity,and neither were control-transfected cells. For TRPV3-expressing cells,camphor (20% of a commercially available stock solution) was used.Camphor by itself did not stimulate eGFP-expressing control N2a cells ornative N2a cells. Stimulation and control protocols were applied as forTRPA1-expressing N2a cells as detailed above in Example 15.

Results are shown in FIG. 24 , which shows the Ca2+ signals for specificstimulation of TRPV1, TRPV2, and TRPV3. Control-transfected N2a cellsdid not respond to capsaicin (TRPV1 activation) or camphor (TRPV3activation). For TRPV1, TRPV2, and TRPV3, Ca2+ transience was notaffected by administration of 16-8 or 16-19. Therefore, compounds 16-8and 16-19 did not affect TRPV1, TRPV2, and TRPV3.

Example 19: Chemical Synthesis of GSK205 Derivatives and Assessment oftheir TRPV4-Inhibitory Potency in Cell-Based Assays

We modified compound GSK205 by generating 7 primary modifications, asshown in FIG. 25 . One additional compound (16-19) that had the combinedrespective modifications of the two most potent compounds, as defined inprimary screens, was also synthesized. We assessed TRPV4-inhibitorypotency of these synthetic compounds in a Ca++ imaging assay in neuronal2a (N2a) permanent tissue culture cells with directed expression ofmammalian (rat) TRPV4. TRPV4 channels were stimulated with a selectiveactivator compound, GSK1016790A (GSK101), used at 5 nM. For first roundassessment, all TRPV4-inhibitory compounds were used at 5 μM (FIG. 26A).Compound 16-43C did not inhibit Ca++ influx, and its effect was similarto vehicle control. All other compounds inhibited TRPV4-mediated Ca++influx, with compounds 16-8 and 16-18 emerging as the two most potent.Compound 16-19 which incorporated the modifications of both 16-8 and16-18, was also effective in inhibiting TRPV4-mediated currents.However, we did not find a significant difference between com-pound16-19 and 16-8, both of which virtually eliminated Ca++ influx.

We then conducted more detailed dose-response assessments for compounds16-8, 16-18 and 16-19, which showed an IC₅₀ of 0.45, 0.81 and 0.59 μM,respectively, vs. an IC₅₀ of 4.19 μM for parental compound GSK205. Thesefindings represent an increased potency of the GSK205-derivativecompounds by approximately 10-fold for 16-8, 8-fold for 16-19 and 5-foldfor 16-18. Surprisingly, compound 16-19 was not significantly morepotent than 16-8, whereas 16-8 was more potent than 16-18. Based onthese results, we tested 16-8 and 16-19 vs GSK205 (as a control) inpatch-clamp studies (FIG. 27 ). Our results indicate significantlyincreased potency of compounds 16-8 and 16-19 as compared to theparental molecule, GSK205 (all applied at 5 μM) in attenuatingTRPV4-mediated currents.

We next decided to assess potency of the most potent compound 16-8 vs.parental compound GKS205 in two types of primary cells that are known toexpress TRPV4 and in which TRPV4 function has been demon-strated in arelevant biological context. We examined articular chondrocytes, whichhave prominent TRPV4 expression, where TRPV4 serves as themechanotransducer of physiologic mechanical loads to regulate the cells'anabolic response, and thus tissue homeostasis, in cartilage¹⁹. Inaddition, we studied brain astrocytes, where TRPV4 expression andrelevant function has been previously demonstrated in regulatingastrocyte cellular edema, in the coupling of neuronal activity tocerebral blood flow, and in mediating CNS traumatic injury³⁵⁻³⁷.Fulfilling our main objective, in both cell types we observedsignificantly increased potency of com-pound 16-8 as compared to theparental molecule, GSK205 (FIG. 28 ). Evaluation of the inhibitorypotency of GSK205 derivatives in these primary cells, which expressfunctional and biologically-relevant TRPV4 without directedover-expression of the channel (FIG. 28 ), directly corroborate thefindings of more basic studies using heterologously TRPV4-overexpressingimmortalized cell lines (FIG. 26 ), strongly supporting our conclusionson the increased potency of the newly derived compounds. Taken together,we identified compound 16-8 as a TRPV4-inhibitory compound withsub-micromolar potency in heterologous systems, with approximately a 10×increase in potency as compared to its parental molecule, GSK205.Moreover, 16-8 proved more effective in TRPV4-expressing primaryskeletal and CNS-derived cells. However, the rational modification tocompound 16-19, intended to further enhance potency, did not yield theintended effect.

Before testing these compounds in relevant in-vivo animal models, wenext tested the cellular toxicity as well as the specificity of thesecompounds against other selected TRP ion channels.

Example 20: Novel TRPV4 Inhibitors are Selective, with Benign ToxicityProfile, Yet Display Potent Inhibi-tion of TRPA1

In heterologously transfected permanent N2a cells, we did not observeinhibitory potency of compounds 16-8 or 16-19 toward TRPV1, TRPV2 andTRPV3 (FIG. 29A). However, we made the unexpected discovery ofsub-micromolar inhibitory potency vs TRPA1 for compounds 16-8 and 16-19,micromolar potency for GSK205, and, remarkably, no significant activityfor compound 16-18 (FIG. 29B). We recorded IC₅₀ of 0.41, 0.43, 5.56 μMand >25 μM for compounds 16-8, 16-19, GSK205 and 16-18, respectively.

In terms of cellular toxicity, we found first evidence of toxicity usinga sensitive cell viability assay over a time-course of 2 days, at 20 μM,and more pronounced effects at 40 μM (FIG. 30 ).

Taken together, our results indicate that compounds 16-8 and 16-19 alsoinhibit TRPA1 at sub-micromolar potency, and their cellular toxicity vstheir inhibitory potency against TRPV4/TRPA1 ranges at a factor of50-100.

Assessment of specificity of 16-8, 16-18 and 16-19 against a widerspectrum of receptors and ion channels will be the subject of dedicatedfuture studies directed toward translation of these compounds to theclinic.

We next evaluated these compounds to an in-vivo model of irritant painknown to rely on both, TRPV4 and TRPA1.

Example 21: TRPV4/TRPA1 Dual-Inhibitors are Effective in ContainingTrigeminal Irritant Pain

We have previously described TRPV4 as a cellular receptor forformalin¹³, and demonstrated its involvement in forma-linirritant-evoked pain behavior, with a focus on trigeminally-mediatedirritant pain behavior. In this recent study, we also demonstrate theco-contribution of TRPV4 together with TRPA1 in trigeminalformalin-evoked pain. We also showed the effective attenuation oftrigeminal formalin-evoked pain behavior using GSK205 in adose-dependent manner. Moreover, we found irritant pain behavior inresponse to selective activation of TRPV4 in the trigeminal territory,which was blocked by GSK205, and the absence of such an effect inTrpv4^(−/−) pan-null mice.

With this pertinent background as a rationale, we applied TRPV4/TRPA1dual-inhibitory compounds 16-8 and 16-19 systemically, using GSK205 ascontrol, at 10 mg/kg dosage. We prioritized the dual-inhibitors overtesting of compound 16-18 (TRPV4-only inhibitor) because (i) thetrigeminal formalin pain model relies on both TRPV4 and TRPA1, (ii) weintended to develop TRPV4/TRPA1 dual-inhibitory molecules towardtranslational use in the first place. None of the compounds wereeffective at significantly diminishing pain behavior in the early phaseafter formalin whisker-pad injection, which represents an acute chemicaltissue injury pain. In the delayed phase, which is understood asneurally-mediated pain indicative of early maladaptive neuralplasticity, there was a significant attenuation of formalin-evoked painbehavior in response to compound 16-8 and 16-19, with compound 16-8diminishing pain behavior at a remarkable >50%¹³, and compound 16-19also showing a robust effect (FIG. 31A,B). Of note, at 10 mg/kg systemicapplication, there was no significant effect of GSK205, which waseffective previously in a dose-dependent manner when applied byintradermal injection¹³. Thus, compounds 16-8 and 16-19, upon systemicapplication, effectively attenuate the late, neurally-mediated phase oftrigeminal formalin pain, and these compounds are more potent in-vivothan their parental compound, GSK205.

In view of these in-vivo findings, taken together with the results fromheterologous cellular expression sys-tems that indicate an additionalTRPA1-inhibitory effect of compounds 16-8 and 16-19, we decided toassess effectiveness of these compounds in a setting ofgenetically-encoded absence of Trpv4 (Trpv4^(−/−) mouse), in order tobetter define their TRPA1-inhibitory potency in-vivo. We observedsignificant residual irritant-pain behavior in all phases of theformalin model in Trpv4^(−/−) mice, consistent with our previousreport¹³ (FIG. 31C,D). Immediate-phase pain behavior was virtuallyeliminated with compounds 16-8 and 16-19, both applied again at 10 mg/kgbody-weight. Late-phase pain behavior was strikingly reduced whenapplying compound 16-8, and still significantly reduced vsvehicle-treated Trpv4^(−/−) when applying compound 16-19, although notas potently as 16-8. A reference TRPA1-inhibitory compound, A967079, wasused at 25 mg/kg body weight as a positive control to inhibit TRPA1,based on a previous report³⁸. Reduction of pain behavior in Trpv4^(−/−)mice was striking, more than 50% in the late neural phase. We notedequal potency of compound 16-8 at 10 mg/kg body weight vs. refer-enceTRPA1-inhibitory compound A967079 at 25 mg/kg body weight, both reducingformalin-evoked trigeminal pain behavior to similarly robust degree(FIG. 31C,D). We conclude that compound 16-8 also functions as a potentTRPA1-inhibitor in an in-vivo irritant pain model specifically designedto assess the contribution of TRPA1 to trigeminal irritant pain, and atleast as potent as an established reference TRPA1-antagonistic compound.

Example 22: Potent TRPV4/TRPA1 Dual-Inhibitor, 16-8, is Effective atControlling Inflammation and Pain in Acute Pancreatitis

These findings define compound 16-8 as a potent TRPV4/TRPA1dual-inhibitor molecule, based on cell-based and live-animal results. Wetherefore decided to test it in a more specific preclinical pain modelthat relies on both, TRPV4 and TRPA1, in order to establishproof-of-principle that a dual-inhibitor can effectively treat pain andinflammation in pancreatitis. We used a pancreatitis model because itprovides high translation potential due to unmet clinical need for neweffective treatments in this condition³⁹, as well as the known role ofboth TRPV4 and TRPA1 in pancreatitis pain and inflammation^(8,25,40).

Pancreatitis was induced with caerulein, a well-characterized model foracute pancreatitis⁴¹. Animals were treated with 10 mg/kg bw 16-8 byintraperitoneal injection, 30 min before induction of inflammation. Wefound significant attenuation of inflammatory parameters, namely edema,which was virtually eliminated in 16-8 treated animals. Furthermore,serum amylase, a marker of inflammatory injury of the pancreas, wassignificantly reduced by 16-8 treatment, as was myelo-peroxidase contentof the pancreas, as a marker of inflammatory cell infiltration of thepancreas. The histopathological score for pancreas inflammation was alsosignificantly reduced (FIG. 32A-E). Of note, pain behavior, similar tothe effect of 16-8 on pancreas edema, was virtually eliminated upontreatment with compound 16-8. Thus, compound 16-8 was found to be highlyeffective in attenuating pain and inflammation of acutechemically-evoked pancreatitis.

Example 23: Benign Preliminary Toxicity and Pharmacokinetics of NovelTPRV4/TRPA1 Inhibitors

Promising properties of potent 16- . . . compounds prompted us toattempt to define their initial preliminary features in terms of in-vivotoxicity and pharmacokinetics, which will be followed by more extensiveand in-depth investigations in future studies. For this, we chosecompound 16-8 as the all-around most potent dual TRPV4/TRPA1 inhibitor,and also compound 16-19 with its potentially increased lipophilicity(TABLE 5). We measured compound concentration in several organs andplasma, and detected micromolar/submicromolar concentrations in liverand kidney, less than 100 nM concentrations in heart, brain, brainstem,trigeminal ganglion and skin. Of note, compounds were virtuallyundetectable in plasma (FIG. 33A). We detected higher concentrations of16-19 in non-nervous tissue, especially liver and kidney, a patternperhaps related to compound 16-19's increased lipo-philicity. Based onthis finding, we next examined 6 and 24 h time-points and detected 10-20fold higher concen-trations of 16-19 at the 6 h time-point, compared tothe 1 h time-point, indicative of compound sequestration into solidorgans (FIG. 33B-D). Values at the 24 h time-point were lower than atthe 6 h but higher than at the 1 h time-point, indicating ongoingprotracted compound clearance. We next confirmed that low/non-detectableconcentrations in plasma were not caused by compounddenaturation/degradation in plasma, as indicated in FIG. 33E, whichshows no loss of detectable compound after 4 h incubation in plasma at37° C., conducted using compound 16-19.

TABLE 5 Properties of GSK205, 16-8, and 16-19. Compound MW PSAc-logP(oct/H2O) GSK205 400 37.53 5.35 16-8 399.6 24.4 6.33 16-19 413.624.2 6.58 MW = Molecular weight. PSA = Polar surface area.C-logP(oct/H2O) indicates calculated logP(octanol solubility/H2Osolubility).

We next performed basic preliminary in-vivo toxicity studies forcompounds 16-8 and 16-19, both at 10 mg/kg bw, which was the effectiveconcentration in both in-vivo pain models. We did not detect firstevidence of cardiac, hepatic and renal toxicity, when comparingcompounds 16-8 and 16-19 with vehicle (FIG. 34 ). For cardiacassessment, we did not detect differences and changes in heart rate over1 h, conducted by EKG at the 6 h time-point. Serum creatinin as markerof renal function and alanine-amino-transferase (ALT) as marker ofhepato-cellular integrity were not significantly elevated in animalstreated with compounds 16-8 or 16-19. Thus, initial evidence for potent16- . . . compounds highlights their acceptable preliminarypharmacokinetics proper-ties as well as lack of gross systemic toxicity.Future studies will be necessary for more detailed assessment of thepharmacokinetics and toxicity of these compounds.

The foregoing description of the specific aspects will so fully revealthe general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific aspects, without undueexperimentation, without departing from the general concept of thepresent disclosure. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed aspects, based on the teaching and guidance presented herein.It is to be understood that the phraseology or terminology herein is forthe purpose of description and not of limitation, such that theterminology or phraseology of the present specification is to beinterpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present disclosure should not be limited byany of the above-described exemplary aspects, but should be defined onlyin accordance with the following claims and their equivalents.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

For reasons of completeness, various aspects of the invention are setout in the following numbered clauses:

Clause 1. A composition comprising an inhibitor and iodine, wherein theinhibitor inhibits TRPV4, TRPA1, or a combination thereof.

Clause 2. A composition comprising an inhibitor and an anesthetic,wherein the inhibitor inhibits TRPV4, TRPA1, or a combination thereof.

Clause 3. The composition of clause 1 or 2 wherein the inhibitor doesnot inhibit TRPV1, TRPV2, or TRPV3.

Clause 4. The composition of any one of the above clauses, wherein theinhibitor comprises a compound according to Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring.

Clause 5. The composition of clause 4, wherein the inhibitor comprises acompound selected from the following:

Clause 6. A method of sanitizing a subject, the method comprisingcontacting the subject with the composition of any one of clauses 1 and3-5.

Clause 7. The method of clause 6, wherein the subject is contacted withthe composition for a period of time sufficient to cause a reduction inthe population of microorganisms on the subject.

Clause 8. The method of clause 6 or 7, wherein the composition isadministered to a surface of the subject, wherein the surface isselected from the group consisting of a skin area, a wound, and anulcer.

Clause 9. The method of any one of clauses 6-8, wherein the compositiondisinfects the subject.

Clause 10. The method of any one of clauses 6-8, wherein the compositionsterilizes the subject.

Clause 11. The method of any one of clauses 6-8, wherein the compositionhas antibacterial activity.

Clause 12. A method of anesthetizing a subject, the method comprisingadministering to the subject the composition of any one of clauses 2-5.

Clause 13. A method of anesthetizing a subject, the method comprisingco-administering an anesthetic and an inhibitor to the subject, whereinthe inhibitor inhibits TRPV4, TRPA1, or a combination thereof.

Clause 14. The method of clause 13, wherein the composition sanitizesand reduces pain.

Clause 15. A method of treating and/or preventing a dermatologicaldisorder in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a TRPV4 and/or TRPA1inhibitor.

Clause 16. The method of clause 15, wherein the dermatological disorderis selected from the group consisting of pancreatitis, epilepsy,arthritis, osteoarthritis, multiple sclerosis, stroke, CNS autoimmunecondition, traumatic brain injury, spinal cord injury, brain edema, CNSinfection, neuro-psychiatric disorder, skeletaldegenerative-inflammatory disorder, trigeminal pain, colitis, andsclerosis.

Clause 17. The method of clause 16 wherein the trigeminal pain comprisesheadache.

Clause 18. The method of any one of clauses 15-17, wherein the TRPV4and/or TRPV4 inhibitor comprises a compound according to Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring.

Clause 19. The method of clause 18, wherein the TRPV4 and/or TRPA1inhibitor comprises a compound selected from the following:

Clause 20. The method of clause 18 or 19, wherein the compound inhibitsTRPV4 and TRPA1.

Clause 21. The method of clause 18 or 19, wherein the compound does notinhibit TRPV1, TRPV2, or TRPV3.

SEQUENCES

(SEQ ID NO: 1) 5′-CCTGCTGGTCACCTACATCA (SEQ ID NO: 2)5′-CTCAGGAACACAGGGAAGGA

The invention claimed is:
 1. A composition comprising an inhibitor andiodine, wherein the inhibitor inhibits TRPV4, TRPA1, or a combinationthereof, wherein the inhibitor comprises a compound according to FormulaI:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring.
 2. Acomposition comprising an inhibitor and an anesthetic, wherein theinhibitor inhibits TRPV4, TRPA1, or a combination thereof, wherein theinhibitor comprises a compound according to Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring.
 3. Thecomposition of claim 1, wherein the inhibitor does not inhibit TRPV1,TRPV2, or TRPV3.
 4. The composition of claim 2, wherein the inhibitordoes not inhibit TRPV1, TRPV2, or TRPV3.
 5. A method of sanitizing aportion of the skin of a subject and reducing associated pain, themethod comprising contacting the portion of the skin of the subject witha composition comprising iodine and a pain inhibitor, wherein the paininhibitor inhibits TRPV4 and TRPA1 and comprises a compound according toFormula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring.
 6. Themethod of claim 5, wherein the compound is selected from the following:


7. The method of claim 5, wherein the portion of the skin of the subjectis contacted with the composition for a period of time sufficient tocause a reduction in the population of microorganisms on the portion ofthe skin of the subject.
 8. The method of claim 5, wherein the portionof the skin of the subject comprises a wound or an ulcer.
 9. The methodof claim 5, wherein the composition disinfects the portion of the skinof the subject.
 10. The method of claim 5, wherein the compositionsterilizes the portion of the skin of the subject.
 11. The method ofclaim 5, wherein the composition has antibacterial activity.
 12. Amethod of anesthetizing a subject, the method comprising administeringto the subject the composition of claim
 2. 13. A method of anesthetizinga subject, the method comprising co-administering an anesthetic and aninhibitor to the subject, wherein the inhibitor inhibits TRPV4, TRPA1,or a combination thereof, wherein the TRPV4 and/or TRPV4 inhibitorcomprises a compound according to Formula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring.
 14. Themethod of claim 13, wherein the composition sanitizes and reduces pain.15. A method of treating and/or preventing a dermatological disorder ina subject in need thereof, the method comprising administering to thesubject an effective amount of a TRPV4 and/or TRPA1 inhibitor, whereinthe TRPV4 and/or TRPV4 inhibitor comprises a compound according toFormula I:

wherein A, B, and C are independently selected from the group consistingof aromatic, heteroaromatic, cycloalkenyl, and heterocycloalkenylgroups; D is C₁-C₃ alkylene; E is a bond, or C₁-C₂ alkylene; and R isselected from the group consisting of hydrogen, hydroxyl, amino, alkyl,alkenyl, heteroalkyl, aromatic ring, or heteroaromatic ring.
 16. Themethod of claim 15, wherein the dermatological disorder is selected fromthe group consisting of pancreatitis, epilepsy, arthritis,osteoarthritis, multiple sclerosis, stroke, CNS autoimmune condition,traumatic brain injury, spinal cord injury, brain edema, CNS infection,neuro-psychiatric disorder, skeletal degenerative-inflammatory disorder,trigeminal pain, colitis, and sclerosis.
 17. The method of claim 16wherein the trigeminal pain comprises headache.
 18. The method of claim15, wherein the TRPV4 and/or TRPA1 inhibitor comprises a compoundselected from the following:


19. The method of claim 15, wherein the compound inhibits TRPV4 andTRPA1.
 20. The method of claim 5, wherein the compound does not inhibitTRPV1, TRPV2, or TRPV3.
 21. The method of claim 5, wherein the iodine isadministered to the subject in a dose of about 0.1 mg to about 2000 mg.22. The method of claim 5, wherein the iodine is administered to thesubject in a dose of about 0.5 to about 60 mg.
 23. The method of claim5, wherein the iodine is present in the composition as elemental iodine.24. The method of claim 5, wherein the iodine is present in thecomposition as an iodine salt.
 25. The method of claim 24, wherein theiodine is present in the composition as potassium iodide and/or sodiumiodide.